Novel aliphatic acyltransferase genes

ABSTRACT

A protein which has an amino acid sequence shown by SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29, or any of these amino acid sequences modified, and which has activity for transferring an aliphatic acyl group to a glycosyl group at the 5-position of flavonoids, and a gene encoding the same.

FIELD OF THE INVENTION

[0001] The present invention relates to a gene encoding a protein which has an activity to transfer an aliphatic acyl group to a glycosyl group at the 5-position of flavonoids, and to a method of using same.

RELATED ART

[0002] In the floriculture industry, it is important to develop new and different varieties of flowering plants. In particular, flower colour is one of the most important characteristics of flowering plants, and classical breeding techniques that rely on crossing have been used extensively to develop new varieties exhibiting various colours. However, since genetic resources are very limited for a particular plant species in which crossing can be carried out, it is rare for a single plant species to have a full spectrum of colour varieties.

[0003] Flower colour is predominantly due to a class of compounds, generally called anthocyanins, which belong to flavonoids. It has been known that there are various anthocyanins in plants, and the molecular structure of many of these compounds have already been determined. The colour of an anthocyanin is determined mainly by its structure (Harborne (1986) The Flavonoids, p. 565). Research has been conducted on enzymes, and genes encoding these enzymes, involved in biosynthesis of anthocyanins. There are instances, for example, in which techniques in molecular biology were applied, and genes were introduced into plants to alter flower colours (Holton et al. (1995) Plant Cell, 7, p. 1071; Tanaka et al. (1998) Plant Cell Physiol. 39. p. 1119). The biochemical pathway for biosynthesis of anthocyanins up to anthocyanidin 3-glucosides is common in most flowering plats (Holton et al. (1995) Plant Cell, 7, p. 1071). Thereafter, anthocyanidin 3-glucosides present in plants are subjected to diverse modifications specific to species or varieties. The diversity of this modification is one of the causes for the diversity of flower colours.

[0004] Although anthocyanins are unstable compounds in neutral solution, their stability is improved by modification with a glycosyl or an acyl group (Forkmann (1991) Plant Breeding, 106, p.1). The colour of anthocynins becomes a little reddish by glycosylation, and becomes blue when an aromatic acyl group is added (Forkmann (1991) Plant Breeding, 106, p1). The acyl groups are broadly divided into the aromatic acyl groups (for example, the caffeoyl group, the coumaroyl group, etc.) and the aliphatic acyl groups (for example, the malonyl group, the acetyl group, etc.). The physiological role of the aliphatic acyl group as concerns flower colour is not known, except that it increases the solubility of anthocyanins.

[0005] Several studies have been reported on purification and biochemical properties of enzymes having an activity to transfer an aliphatic acyl group to anthocyanins [Archives of Biochemistry and Biophysics, 1981, 208, 233-241 (Crude purification, molecular weight, and examination of substrate-specificity of Flavonol 3MaT (an enzyme catalyzing the reaction of transferring a malonyl group to a glycosyl group at the 3-position of a flavonol) and Flavone/Flavonol 7 MaT (an enzyme catalyzing the reaction of transferring a malonyl group to a glycosyl group at the 7-position of flavone and flavonol) of parsley); Archives of Biochemistry and Biophysics, 1983, 224, 261-271 (Measurement of activity of flavonol 3MaT and Flavone/Flavonol 7 MaT in various organs of parsley); Archives of Biochemistry and Biophysics, 1983, 226, 206-217 (Purification into single sample of Flavonol 3MaT and Flavone/Flavonol 7 MaT of parsley, and Preparation of 3MaT antibody); Eur. J. Biochem. 1983, 133, 439-448 (Confirmation of existence and structure of malonylated apigenin 7-0-glucoside in parsley by means of NMR etc.); Archives of Biochemistry and Biophysics, 1984, 234, 513-521 (Determination of optimum pH, molecular weight and Km of 7MaT for an isoflavone of a pea); Phytochemistry, 1993, 32, 1425-1426 (Confirmation of existence of aliphatic acyl transferase activity to cyanidin 3-glucoside in the crude extract from flower petals of Dendranthema morifolium, a plant belonging to Asteraceae); Plant Science, 1996, 118, 109-118 (Confirmation of malonyl transferase activity in crude extract from cultured cells of Ajuca reptans); Phytochemistry, 1999, 52, 15-18 (Determination of substrate specificity of malonyl transferase derived from flower petals of dahlia)]. However, the primary structures of the proteins has not been determined, nor has cloning of the gene been reported.

DISCLOSURE OF THE INVENTION

[0006] It is an object of the present invention to determine an effect of malonylation among acylation, by an aliphatic acyl transferase for transferring an acyl groups to anthocyanins upon colour of flowers, and to provide a gene which encodes a protein having an activity to transfer an aliphatic acyl group, preferably a gene which encodes a protein having an activity to transfer an aliphatic acyl group to anthocynins. It is possible to alter the colour of flowers by introducing a gene which encodes a protein having an activity to transfer an aliphatic acyl group, in accordance with the present invention, into a plant and by expressing the same.

[0007] As described above, there have been no reports on the effect of malonylation of anthocyanins upon flower colour. In order to determine this effect, the colour of three solutions of anthocyanins, that is, delphinidin 3,5-diglucoside, awobanin(delphinidin 3-(coumaroyl) glucoside-5-glucoside), malonyl-awobanin(delphinidin 3-(coumaroyl) glucoside-5-(malonyl)glucoside), was compared, and it was found that the colour of malonyl-awobanin is the bluest, indicating that malonylation causes anthocyanins to become bluer.

[0008] Thus, purification of a malonyl transferase was attempted using flowers of salvia as the material. Then, a partial amino acid sequences of the purified protein were determined, and based on this information, a DNA fragment of the gene encoding the malonyl transferase of salvia was amplified using the PCR method. Using this DNA fragment as a probe, the cDNA library of flower of Salvia guaranitica was screened, and two genes encoding malonyl transferase were obtained. Further, using these genes as probes, homologs were obtained from Salvia splendens, perilla, and lavender.

[0009] Therefore, according to the present invention, there is provided a gene encoding a protein which has an amino acid sequence according to any one of SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29, and has an activity to transfer an aliphatic acyl group to a glycosyl group at the 5-position of flavonoids, or a gene encoding a protein which has any of these sequences modified by addition or deletion of one or more amino acids and/or substitution by other amino acids, and has activity for transferring an aliphatic acyl group to a glycosyl group at 5-position of flavonoids.

[0010] According to the present invention, there is also provided a gene encoding a protein which has an amino acid sequence exhibiting homology of 50% or more with any of the sequences according to SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29, and has an activity to transfer an aliphatic acyl group to a glycosyl group at the 5-position of a flavonoids.

[0011] Further, according to the present invention, there is provided a gene which hybridizes to a part or all of the nucleotide sequences encoding any of the amino acid sequences according to SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29 under the condition of 5×SSC, 50° C., and which encodes a protein having an activity to transfer an aliphatic acyl group to a glycosyl group at 5-position of flavonoids.

[0012] The present invention additionally provides a vector comprising the above-described gene.

[0013] The present invention also provides a host transformed by the above-described vector.

[0014] The present invention further provides a protein encoded by any of the above-described genes.

[0015] The present invention also provides a method of making the protein, comprising the steps of: culturing, or growing, the above-described host; and collecting, from the host, the protein having an activity to transfer an aliphatic acyl group to a glycosyl group at 5-position of flavonoids.

[0016] The present invention further provides a transgenic plant having the above-described gene introduced, or offspring of the plant or tissue thereof having the same property.

[0017] The present invention further provides a cut flower of the above-described plant or offspring thereof having the same property.

[0018] The present invention further provides a method of altering flower colour using the above-described gene.

[0019] The present invention further provides a method of making a flower blue by using the above-described gene.

PREFERRED EMBODIMENTS OF THE INVENTION

[0020] Genes of the present invention include, for example, those encoding amino acid sequences according to SEQ ID NO: 2, 4, 6, 23, 25, 27 and 29. However, it is known that a protein having an amino acid sequence that is modified by addition or deletion of plural amino acids and/or by substitution of other amino acids, exhibits the same enzyme activity as the original protein. Therefore, a protein having an amino acid sequence according to any one of SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29 modified by addition or deletion of one or more amino acids and/or by substitution by other amino acids, and a gene encoding the protein, is within the scope of the present invention, as long as the protein has an activity to transfer an aliphatic acyl group to a glycosyl group at the 5-position of flavonoids.

[0021] The present invention also relates to a gene which hybridizes with a nucleotide sequence encoding amino acid sequences according to any one of SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29, or a part of the nucleotide sequences, preferably a nucleotide sequence encoding 6 or more amino acids, for example, 6 or more amino acids in a concensus region, under the condition of, for example, 5×SSC, at 50° C., and which encodes a protein having an activity to transfer an aliphatic acyl group to a glycosyl group at the 5-position of flavonoids. A suitable hybridization temperature varies with the nucleotide sequence or the length thereof, and a temperature of 50° C. or lower is preferred when the probe is a DNA fragment of 18 bases encoding 6 amino acids.

[0022] Genes selected by such hybridization include naturally-occurring genes, including, but not limited to, genes derived from plants such as genes derived from petunia, torenia, etc. Also, a gene selected by hybridization may be either cDNA, or genome DNA.

[0023] Further, the present invention also relates to the use for altering flower colour, of a gene encoding a protein which has an amino acid sequence having a homology of about 50% or more, preferably 60% or 70% or more, further preferably 80% or 90% or more, with the amino acid sequence according to any one of SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29, and which has an activity to transfer an aliphatic acyl group to a glycosyl group at the 5-position of flavonoids.

[0024] A gene having a native nucleotide sequence can be obtained, for example, by screening of a cDNA library, as shown more specifically in Examples. A DNA encoding a protein having a modified amino acid sequence can be synthesized starting from DNA having a native nucleotide sequence by means of usual methods such as a site-specific mutagenesis or the PCR method. For example, a DNA fragment to which a desired modification is to be introduced is first obtained by treatment of a native cDNA or genome DNA with restriction enzymes. Then, using this as a template, site-specific mutagenesis or the PCR method is performed with a primer having the desired mutation introduced therein, to obtain a DNA fragment having the desired modification introduced therein. Thereafter, the DNA fragment having the desired modification introduced therein may be ligated to a DNA fragment encoding other portions of the target protein.

[0025] Alternatively, in order to obtain DNA encoding a protein which has a shortened amino acid sequence, DNA encoding an amino acid sequence longer than the target amino acid sequence, such as the DNA encoding the full-length amino acid sequence, may be cut with suitable restriction enzymes. If the resulting DNA fragment does not encode the entire target amino acid sequence, a DNA fragment consisting of the missing sequence may be synthesized and ligated.

[0026] By expressing the obtained gene using a gene expression system in Escherichia coli and yeast, and by measuring the enzyme activity, it is possible to confirm that an obtained gene encodes a protein having an activity to transfer an aliphatic acyl group. It is also possible by expressing the gene to obtain, as a gene product, a protein having an activity to transfer an aliphatic acyl group. It is also possible by using an antibody to an amino acid sequence according to SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29, to obtain a protein having an activity to transfer an aliphatic acyl group. Further, it is possible to use an antibody to clone a gene encoding a protein having an activity to transfer an aliphatic acyl group derived from other living organisms.

[0027] Therefore, the present invention relates to a recombination vector, especially an expression vector, comprising above described gene, and to a host transformed by the vector. Both procaryote and eukaryote may be used as a host. In procaryote, bacteria such as Escherichia coli that belongs to the genus Escherichia, or Bacillus subtilis that belongs to the genus Bacillus may be used as a usual host. As a eukaryotic host, a lower eukaryote, for example, a eukaryotic micro-organism such as yeast and fungi which belong to the fungi may be used.

[0028] In yeast, a micro-organism belonging to the genus Saccharomyces such as Saccharomyces cerevisiae or the like may be used as a host. In mold fungi, a micro-organism belonging to the genus Aspergillus such as Aspergillus orvzae, Aspergillus niger, and a micro-organism belonging to the genus Penicillium, may be used as hosts. Animal cells and plant cells may also be used as hosts. In animal cells, cell systems derived from a mouse, hamster, monkey, a human, etc. may be used. Insect cells such as silkworm cells, or even an adult silkworm itself, may be used as a host.

[0029] The expression vector of the present invention includes expression control regions depending upon the kind of host to which it is to be introduced, such as a promotor and a terminator, replication origin, and the like. As a promotor for an expression vector in bacteria, commonly used promotors such as a trc promotor, tac promotor, lac promotor or the like may be used. As a promotor for an expression vector in yeast, glyceraldehyde-3-phosphate dehydrogenase promotor, pH05 promoter or the like, and as a promotor for an expression vector in fungi, an amylase promotor, a trpC promotor or the like may be used. As a promotor for an expression vector in animal cell hosts, a viral promotor such as SV 40 early promotor, SV 40 late promotor, or the like may be used. Construction of the expression vector may be performed in accordance with any of the usual methods known to those skilled in the art using restriction enzymes, ligases, etc. Transformation of host cells with the expression vector may also be performed in accordance with any of the usual methods.

[0030] The target protein can be obtained by culturing, raising or growing the host transformed with the above described expression vector, and by recovering a gene product from the culture or the like, and purifying in accordance with usual methods, for example filtration, centrifuging, disruption of cells, gel filtration chromatography, ion exchange chromatography, and the like.

[0031] The present invention is not limited to a gene derived from salvia and encoding a protein which has an activity to transfer an aliphatic acyl group. The present invention relates to use of a gene encoding a protein which has activity for transferring an aliphatic acyl group. The origin of the protein having an activity to transfer an aliphatic acyl group may be plants, animals, or microorganisms. Irrespective of the origin, such a protein can be equally applied to alteration of flower colour, as long as it has activity for transferring an aliphatic acyl group. Further, the present invention relates to a transgenic plant or its offspring or tissue thereof, including cut flowers, which is obtained by introducing a gene encoding a protein having activity to transfer an aliphatic acyl group, and which has its hue thereby modified.

[0032] By using the gene obtained according to the present invention, which encodes a protein having an activity to transfer an aliphatic acyl group, anthocyanins accumulated in vacuoles can be acylated so as to become blue, and as a result, flower colour can be altered to blue. In the present state of the art, it is possible to introduce a gene into a plant and to cause the gene to be expressed in a constructive or tissue-specific fashion. It is also possible to suppress the expression of a target gene using, for example, an anti-sense method or a co-suppression method.

[0033] Examples of plants that can be transformed in this manner include, but are not limited to, roses, chrysanthemums, carnations, snapdragons, cyclamens, orchids, lisianthus, freesias, gerberas, gladiolus, gypsophila, kalanchoes, lilies, pelargoniums, geraniums, petunias, torenias, tulips, rice, barley, wheat, rapeseed, potatos, tomatos, poplars, bananas, eucalyptuses, sweet potatos, soybeans, alfalfa, lupine, and corn.

EXAMPLES

[0034] The present invention will be described in detail below in accordance with Examples thereof. Unless otherwise specified, the molecular biological techniques employed are those set forth in Molecular Cloning (Sambrook et al., 1989).

Example 1 Change of Colour of Various Anthocyanins Depending Upon pH

[0035] Delphinidin 3,5-diglucoside, awobanin (delphinidin 3-(coumaroyl) glucoside-5-glucoside), malonyl-awobanin (delphinidin 3-(coumaroyl)glucoside-5-(malonyl) glucoside) were dissolved in McIlvaine buffer (pH 5.3, pH 5.6, pH 6.0) in concentrations of 0.1 mM, 0.3 mM, 0.5 mM, respectively, and colours of the solutions were evaluated using Colour charts (Royal Horticulture Society). Delphinidin 3,5-diglucoside can be obtained from its 3,5-diacetylglucoside form by removing the acetyl portion in alkaline hydrolysis reaction (Tetrahedron, 48, 4313-4326, 1992).

[0036] Awobanin can be obtained from malonyl-awobanin by removing its 5-malonyl portion (Tetrahedron Lett. 24, 4863-4866, 1983). Malonyl-awobanin was obtained by extraction from plants using the method as set forth in Tetrahedron Lett. 24, 4863-4866, 1983. The greater the number in the Colour chart, the bluer the colour is. When the number is the same, symbol A represents the bluest colour. The results of the test are summarized in Table 1. In all concentrations and pH, malonyl-awobanin was the bluest, indicating that the malonyl group caused anthocyanins to become blue. TABLE 1 Number Number in Colour Number in in Colour chart Colour chart chart Anthocyanins Concentration at pH 5.3 at pH 5.6 at pH 6.0 Delphinidin 0.1 mM 84C 84C too faint to 3,5- be measured diglucoside Delphinidin 0.3 mM 85A 86D 88C 3,5- diglucoside Delphinidin 0.5 mM 86D 90B 88B/C 3,5- diglucoside Awobanin 0.1 mM 85C 85A 91B Awobanin 0.3 mM 86D not tested 91A Malonyl- 0.1 mM 85C 91B 91B awobanin Malonyl- 0.3 mM 92A 93B 96C awobanin Malonyl- 0.5 mM 93B not tested 96A awobanin

Example 2 Measurement of Activity of Malonyl Transferase of Salvia

[0037] Measurement of activity of malonyl transferase was conducted using reaction solution 100 μl (potassium phosphate of final concentration of 20 mM, pH 7.0, containing shisonin 10 μg, malonyl CoA 10 μg, and an enzyme sample to be measured, dissolved in 0.01% trifluoro acetic acid). After reaction was carried out at 30° C. for 20 minutes, the reaction was terminated by adding 200 μl of 0.05% TFA aqueous solution cooled on ice. Quantification of shisonin and malonyl-shisonin was conducted by reverse phase high performance liquid chromatography (DYNAMAX HPLC system) using a Shodex Asahipak ODP-50 4E column, and using a linear concentration gradient with 0.5% TFA solution as the solution and 0.5% TFA, 50% acetonitrile aqueous solution as the solution, such that concentration of the B solution was 45%, 45%, 55%, 100%, 100%, 45%, and 45%, at time 0, 3, 17, 18, 23, 24, and 30 minutes, respectively, after the start of separation, at a flow rate of 0.7 ml/min, using 50 μl of reaction solution, monitoring absorption at 520 nm.

Example 3 Protein Purification of Malonyl Transferase of Salvia

[0038] Purification of malonyl transferase was conducted using red flowers 2,644 g of Salvia splendens as starting material. Flowers of salvia were collected in its entirety including calyces immediately before blossom, and were stored at −80° C. until use in experiments.

[0039] To 500 g of salvia flowers, polyvinylpolypyrrolidone (PvPP) 100 g, extraction buffer (100 mM potassium phosphate (pH 7.0), 30 mM 2-mercaptoethanol, 5 mM EDTA) 3 L and phenylmethylsulfonylfluoride (PMSF) of a final concentration 0.5 mM were added, and powdered with a HEAVY DUTY BLENDER (WARING). The solution containing the powdered material was centrifuged at 7,500×G for 20 minutes. Supernatant was filtered under reduced pressure (filter paper, Whatman 114) to obtain a crude enzyme solution. A crude enzyme solution of 10.4 L was obtained from flowers of 2,664 g.

[0040] Next, ammonium sulfate fractionation was conducted, and an enzyme was recovered as precipitation in 20% to 50% saturated ammonium sulfate fraction and dissolved in buffer A (100 mM potassium phosphate (pH 7.0), 30 mM 2-mercaptoethanol, 1 mM EDTA, 0.1 mM PMSF)(2920 ml). 280 ml of Octyl Sepharose Fast Flow (Amersham Pharmacia Biotech Co.) was added to this, and ammonium sulfate was slowly added to a final concentration of 30% saturation while the solution was slowly stirred. After adequate stirring, the solution was allowed to stand still overnight. After confirming that enzyme activity of malonyl transferase was not left in the supernatant liquid, the gel in the sludge was recovered by filtration under reduced pressure (filter paper, Whatman 114). The gel was extensively washed with buffer B (20 mM potassium phosphate (pH 7.0), 30 mM 2-mercaptoethanol, 20% saturated ammonium sulfate), while being filtered under reduced pressure.

[0041] 280 ml of buffer C [20 mM potassium phosphate (pH 7.0), 15 mM 2-mercaptoethanol, 50% ethylene glycol, 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS)] was added to the gel, and after being gently stirred for 30 minutes, a malonyl transferase active fraction was recovered by filtration under reduced pressure. This elution operation with buffer C was repeated 10 times to collect an active fraction. After the active fraction was concentrated using Pellicon (Biomax 8K, MILLIPORE CORPORATION), and was desalted in buffer D (10 mM potassium phosphate (pH 7.0), 15 mM 2-mercaptoethanol, 0.03% TritonX-100), 155 ml of enzyme solution 1 was obtained.

[0042] MIMETIC Yellow 2 (Nacalai Tesque Co.) 100 ml was loaded to Econocolumn (φ1.0 cm×120 cm, Japan Bio Rad Laboratories Co.), and was equilibrated with buffer D. After the total amount of the enzyme solution 1 was applied and washed with buffer D, protein bound to the column was eluted using buffer E (20 mM potassium phosphate (pH 7.0), 30 mM 2-mercaptoethanol, 0.05% CHAPS). The active fraction was concentrated using Pellicon and ultrafiltration (YM-10, MILLIPORE CORPORATION), and 32 ml of enzyme solution 2 was obtained.

[0043] MIMETIC Red 3 (Nacalai Tesque Co.) 50 ml was loaded to Econocolumn (φ1.5 cm×30 cm, Japan Bio Rad Laboratories Co.), and was equilibrated with buffer F (5 mM potassium phosphate (pH 7.0), 15 mM 2-mercaptoethanol, 0.03% TritonX-100). After the total amount of enzyme solution 2 was applied and washed with buffer F, the proteins bound to the column were eluted using buffer G (5 mM potassium phosphate (pH 7.0), 30 mM 2-mercaptoethanol, 0.03% TritonX-100, 0.1 mM acetylCoA). After the active fraction was concentrated using Pellicon, ultrafiltration, and Centricon, it was desalted using buffer H (30 mM potassium phosphate (pH 7.0), 30 mM 2-mercaptoethanol, 0.03% TritonX-100), and 3 ml of enzyme solution 3 was obtained.

[0044] MIMETIC Red 3 (Nacalai Tesque Co.) 5 ml was loaded to Econocolumn (φ1.0 cm×10 cm, Japan Bio Rad Laboratories Co.), and was equilibrated with buffer F. After the total amount of the enzyme solution 3 was applied and washed with buffer F, the proteins bound to the column were eluted using buffer G. After the active fraction was concentrated using Centricon, 1.5 ml of enzyme solution 4 was obtained.

[0045] After a MonoQ5/5 column (Amersham Pharmacia Biotech Co.) was equilibrated with buffer G, the total amount of the enzyme solution 4 was applied at a flow rate of 0.05 ml/min and was washed with buffer F (flow rate 0.05 ml/min for 60 minutes). The proteins bound to the column were eluted using a 0 to 100% linear gradient (flow rate 0.05 ml/min, for 400 min) formed with buffer I (20 mM potassium phosphate (pH 7.0), 30 mM 2-mercaptoethanol, 0.03% TritonX-100, 1 mM NaCl), and after the active fraction was concentrated using Centricon, 0.8 ml of enzyme solution 5 was obtained.

[0046] After Phenyl Superose HR 5/5 (Amersham Pharmacia Biotech Co.) was equilibrated with buffer B, the enzyme solution 5 having ammonium sulfate added to a final concentration of 20% saturation was applied at a flow rate of 0.02 ml/min. After the column was washed with buffer B, the proteins bound to the column were eluted using a 0 to 100% linear gradient formed with buffer C (flow rate of 0.02 ml/min, for 500 minutes), and the active fraction was obtained.

[0047] Further, a preparative electrophoresis system (Bio Phoresis III, Atto Co.) was used for fractionation, and the obtained fraction of 3.2 ml of malonyl transferase was concentrated to 40 μl using ultrafiltration membrane concentration, with substituting with 0.02% SDS/75 mM Tris-HCl buffer (pH 8.8). Thereafter, final purification was performed using a reverse phase column (PorosR2H, Japan Perceptive Co.) HPLC. Separation was conducted under the condition of a linear concentration gradient in 0.1% TFA formed by an acetonitrile concentration from 8% to 80%, at a flow rate of 0.1 ml/min for 60 minutes, and monitoring absorption at 280 nm, only the peak fraction was recovered. Since the protein finally obtained turned out to be a single band of 47 kDa, it was determined that the molecular weight of the malonyl transferase was 47 kDa.

Example 4 Determination of Partial Amino Acid Sequences of the Malonyl Transferase of Salvia

[0048] 2 pmol of trypsin (Promega Co.) was added to the protein that was recovered in Example 3 as a single band. The protein was digested at 37° C. for 30 hours, and determination of the structure of each peptide fragment was attempted. Solution digested by trypsin was separated using reverse phase HPLC (μRPC C2/C18, Amersham Pharmacia Biotech Co.) into each peptide fragment. Separation was conducted under 0.1% trifluoroacetic acid using a linear concentration gradient formed with acetonitrile concentration from 8% to 80% for 60 minutes at a flow rate of 0.1 ml/min, and, while monitoring absorption at 215 nm, only absorption peak fractions were collected.

[0049] Each peak fraction was concentrated and dried by speed-back, and was dissolved in 30 μl of 37% acetonitrile and subjected to analysis using an amino acid sequencer (PSQ-1, Shimadzu Corporation). As a result, amino acid sequences of 12 peptides were obtained. The amino acid sequences are shown below.

[0050] MTT20: Tyr-Ala-Ala-Gly-Asp-Ser-Val-Pro-Val-Thr-Ile-Ala-Ala-Ser-Asn (SEQ ID NO: 7)

[0051] MTT21-1: Leu-Leu-Phe-Tyr-His-His-Pro-Ser-Ser-Lys (SEQ ID NO: 8)

[0052] MTT21-2: Ser-Gly-Asp-Lys-Ser-Asp-Glu-Asn-Ala-Pro-Glu-Leu-Phe-Ile-Ile-Pro-Ala-Asp-Ala (SEQ ID NO: 9)

[0053] MTT22-1: Met-Ala-Ala-Phe-Glu-Glu-Val-Phe (SEQ ID NO: 10)

[0054] MTT23: Trp-Leu-His-Tyr-His-Pro-Val (SEQ ID NO: 11)

[0055] MTT26: Gly-Ala-Glu-Asn-Trp-Met-Ser-Asp-Ile-Phe-Lys (SEQ ID NO: 12)

[0056] MTT27-2: Leu-Ala-Ala-Glu-Xaa-Gly-Phe-Ala-Val-Ala-Ala-Ala-Ala-Ile-Gly-Gly-Gly-Ile-Ile-Gly (SEQ ID NO: 13)

[0057] MTT28: Ser-Phe-Ile-Asn-Asp-Pro-Asn-Lys-Ile-Asp-Ala-Ile-Phe (SEQ ID NO: 14)

[0058] MTT141: Thr-Ala-Ser-Phe-Pro-Leu-Pro-Thr-Asn-Arg (SEQ ID NO: 15)

[0059] MTT141-2: Phe-Pro-Gln-Leu-Arg (SEQ ID NO: 16)

[0060] MT142: Ala-Asp-Phe-Gly-Trp-Gly-Lys (SEQ ID NO: 17)

[0061] MTT291: Asp-Ala-Asp-Gln-Phe-Tyr-Asp-Leu-Leu-Pro-Pro-Ile-Pro-Pro (SEQ ID NO: 18)

Example 5 Amplification of Gene Fragments Encoding Malonyl Transferase of Salvia

[0062] Based on the partial amino acid sequences MTT20 and MT142 obtained in Example 4, the following primers were constructed.

[0063] MTT20-1: 5′-TA(T/C) GCI GCI GGI GA(T/C) TCI GTI CCl GT-31 (I: inosine) (SEQ ID NO: 19)

[0064] MTT20-3: 5′-GTI CCl GTI ACI AT(A/T/C) GCI GC-3′ (SEQ ID NO: 20)

[0065] ATCRr2: 5′-(T/C)TT ICC CCA ICC (A/G)AA (A/G)TC IGC-3′ (SEQ ID NO: 21)

[0066] PCR was carried out using cDNA prepared from salvia flowers as a template, with reactant solution having that following composition in a total amount of 100 μl; 1×TAKARA PCR buffer, 200 mM dNTPs, salvia cDNA 100 ng, MTT20-1 primer 1 pmol/μl, ATCRr2 primer 1 pmol/μl, TAKARA rTaq 2.5 units. Reaction was conducted at 96° C. for 1 minute, followed by 30 cycles with each cycle consisting of 1 minute at 96° C., 2 minutes at 42° C. and 3 minutes at 72° C., and further followed by 7 minutes at 72° C.

[0067] Nested PCR was carried out using this reaction product as a template, and using MTT20-3, ATCRr2 primers, with reactant solution of the same composition as described above. Reaction was conducted at 96° C. for 1 minute, followed by 30 cycles with each cycle consisting of 1 minute at 96° C., 2 minutes at 50° C. and 3 minutes at 72° C., and further followed by 7 minutes at 72° C. The PCR product obtained in this manner was subjected to subcloning, and its sequence was determined. As a result, in the deduced amino acid sequence of the reaction product of about 900 bp, partial amino acid sequences MTT141, MTT26, MTT27-2 (SEQ ID NO: 15, 12 and 13) beside with those used for design of primers were found. Thus, it was evident that the reaction product was a gene fragment encoding a purified protein.

Example 6 Isolation of cDNA Encoding the Malonyl Transferase of Salvia

[0068] A cDNA library derived from flowers of Salvia guaranitica was constructed using the KZAP II directional cDNA synthesis kit of Stratagene Co. in accordance with the method recommended by the manufacturer of the kit. About 200,000 clones of this library were screened using the washing condition (5×SSC, 0.1% SDS, 37° C.) with the DNA fragment of 889 bp obtained in Example 3 as a probe, and 10 clones were finally obtained as positive clones. These clones were classified into three kinds of groups, and the longest clones in these group are named as SgMaT1, SgMaT1′, and SgMaT2, respectively. Screening of the library was conducted in accordance with known methods (for example, Fujiwara et al., 1998, Plant J. 16, 421).

[0069] SgMaT1 and SgMaT1′ are 1419 bp and 1471 bp, respectively, and both lacked initiation methionine. Since SgMaT1 and SgMaT1′ exhibit an identity of 98% at amino acid level, they were considered as an allelic gene encoding the same enzyme. In the deduced amino acid sequence of SgMaT1, all the partial amino acid sequences of purified malonyl transferase determined in Example 4 were confirmed, although some are partially different. The partial difference is probably due to the difference of species of salvia used.

[0070] From these results, it became evident that SgMaT1 and SgMaT1′ genes encode an enzyme for transferring a malonyl group to a glycosyl group at the 5-position of anthocyanins. SgMaT2 has a cDNA of 1530 bp, which includes an open reading frame of 1260 bp encoding the full length. At the amino acid level, SgMaT1 exhibited 52% identify with SgMaT2. All of these genes exhibited a identify of 37 to 47% with acyl transferases of other plants.

[0071] In gentian, acyl transferases having different functions show identify of only 35 to 40%, even in the same varieties (Yonekura-Sakakibara et al., 2000, Plant Cell Physiol. 41: 495-502). Therefore, identity of 55% between SgMaT1 and SgMaT2 suggests, although the fact that the two genes are derived from the same variety is allowed for, that SgMaT1 and SgMaT2 are similar in function, and that SgMaT2 also catalyzes the reaction of transferring a malonyl group to anthocyanins. The nucleotide sequences of SgMaT1, SgMaT1′, and SgMaT2 are shown in SEQ ID NO: 1, 3, and 5, respectively, and the amino acid sequences deduced from these nucleotide sequences are shown in SEQ ID NO: 2, 4, and 6, respectively.

Example 7 Confirmation of Enzyme Activity of the Malonyl Transferase in Escherichia coli

[0072] A single colony of Escherichia coli having plasmid pSgMaT1 (including SgMaT1 gene at EcoRI, XhoI sites of pBluescriptSK⁻ (Stratagene), and capable of expressing an SgMaT1 gene product as a fusion protein with lac Z protein by addition of isopropyl-beta-thiogalactoside (IPTG)) introduced, was inoculated to LB medium containing ampicilin to a final concentration of 50 mg/L, and was precultured overnight while being shaked at 37° C. The preculture (2 ml) was inoculated to LB medium of 100 ml containing ampicilin, and was incubated at 30° C. until absorbance at 600 nm became 0.5. Then, IPTG was added to the culture medium to a final concentration of 1 mM. The culture was further maintained at 30° C. for 9 hours after the addition of IPTG, and then was collected. The collected cells were suspended in a buffer (0.1 M KPB, pH 7.0, 30 mM 2-mercaptoethanol, 1 mM EDTA, 0.1 mM PMSF, 0.1% TritonX-100), and disrupted by ultrasonic treatment while cooled on ice.

[0073] Enzyme activity was measured using a supernatant (soluble fraction) obtained after centrifuging. Escherichia coli containing only pBluescriptSK⁻ was processed in the same manner as control. Measurement of activity was conducted using shisonin as a substrate in accordance with Example 2.

[0074] In the reaction product from the reaction using the pSgMaT1 gene expression product, malonyl-shisonin was detected (Rt 12.2 minutes) in addition to shisonin (Rt 9.7 minutes), while only shisonin was detected in control. It was confirmed from this that the SgMaT1 gene encodes an enzyme having activity of transferring the malonyl group.

Example 8 Isolation of cDNA Encoding Malonyl Transferase Derived From Salvia (2)

[0075] A cDNA library of flower petals of salvia (Salvia splendens) was constructed using a ZAP-cDNA Synthesis Kit (Strategene Co.) with λZAP II (Strategene Co.) as a vector, in accordance with the method recommended by the manufacturer. Using SgMaT1 obtained in Example 6 as a probe, screening was carried out by the method described in Example 6. The clone with the longest cDNA was denoted by SsMaT1. This nucleotide sequence is shown in SEQ ID NO: 22, and the amino acid sequence deduced from this nucleotide sequence is shown in SEQ ID NO: 23. Considering the sequence, this cDNA is not considered to be a full-length cDNA.

[0076] SsMaT1 exhibited 92% identity with SgMat1, and 52% identity with SgMaT2 at the amino acid level.

[0077] Similarly, the same library was screened using SgMaT2 as a probe, and SsMaT2 was obtained. This nucleotide sequence is shown in SEQ ID NO: 24, and the amino acid sequence deduced from this nucleotide sequence is shown in SEQ ID NO: 25.

[0078] SsMaT2 exhibited 53% identity with SgMat1, 96% identity with SgMaT2, and 52% identify with SsMaT1 in amino acid level.

Example 9 Isolation of cDNA Encoding the Malonyl Transferase Derived From Perilla

[0079] Young red leaves were collected from perilla (Perilla frutescens), and starting from this material, a cDNA library was constructed using a ZAP-cDNA Synthesis Kit (Strategene Co.) with λZAP II (Strategene Co.) as a vector, in accordance with the method recommended by the manufacturer. As in Example 8, this library was screened using SgMaT1 as a probe, and the clone having the longest cDNA among obtained clones was denoted by PfMaT1. The nucleotide sequence of PfMaT1 is shown in SEQ ID NO: 26, and the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 27.

[0080] PfMaT1 exhibited 67% identity with SgMaT1, 57% identity with SgMaT2, 65% identity with SsMaT1, and 57% identity with SsMaT2 in amino acid level.

Example 10 Isolation of cDNA Encoding the Malonyl Transferase Derived From Lavender

[0081] A cDNA library of lavender (Lavendula angustifolia) was constructed using a ZAP-cDNA Synthesis Kit (Strategene Co.) with λZAP II (Strategene Co.) as a vector, in accordance with the method recommended by the manufacturer. Using SgMaT2 obtained in Example 6 as a probe, screening was carried out by the method as described in Example 6, and LnMaT2 was obtained. This nucleotide sequence is shown in SEQ ID NO: 28, and the amino acid sequence deduced from this nucleotide sequence is shown in SEQ ID NO: 29.

[0082] LnMaT2 exhibited 53% identity with SgMaT1, 65% identity with SgMaT2, 51% identity with SsMaT1, 64% identity with SsMaT2, and 56% identity with PfMaT1 in amino acid level.

Example 11 Expression of S. splendens MaT1

[0083] A primer for introducing a BamHI site at the 5′-end of the SsMaT1 gene obtained in Example 8 (Primer #1: 5′-GGA TCC ATC GAG GGA CGC ATG ACA ACA ACA ACA AC-3′ (SEQ ID NO: 30)), a primer for introducing a BamHI site at the 3′-end of the SsMaT1 gene (Primer #2: 5′-GGA TCC TTA CAA TGG TTC GAC GAG CGC CGG AGA-3′ (SEQ ID NO: 31)), and a primer for deleting the BamHI site in the SsMaT1 gene (Primer #3: 5′-G GAC CCG CCG ATA CCG GAA AAT TAC TTC-3′ (SEQ ID NO: 32)) were synthesized. The primer #1 was designed such that a Factor Xa cleavage site (Ile-Glu-Gly-Arg) is encoded just before the SsMaT1 initiation codon, methionine.

[0084] A first PCR reaction was carried out using Primer #2 and Primer #3 with the plasmid (pBK-CMV-SsMaT1) having SsMaT1 cDNA introduced at multi-cloning sites (EcoRI, XhoI) of pBK-CMV phagemid vector (Toyobo Co.) as a template (composition of reactant: pBK-CMV-SsMaT1 100 ng, 1xpfu buffer (Stratagene Co.), 200 μM dNTPs, 1 μM Primer #2, 1 μM Primer #3, 2.5 U pfu polymerase (Stratagene Co.); reaction condition: 96° C. 2 minutes, (96° C. 1 minute, 70° C. 1 minute, 72° C. 3 minutes) x30 cycles, 72° C. 7 minutes), and a PCR product (about 500 bp) was obtained.

[0085] Using the double strand DNA fragment of SsMaT1 obtained as the first PCR product and Primer #1, a second PCR reaction was carried out (composition of reactant: pBK-CMV-SsMaT1 100 ng, 1xpfu buffer, 200 μM dNTPs, 1 μM Primer #1, the first PCR product 100 ng, 2.5 U pfu DNA polymerase (Stratagene Co.); reaction conditions: 96° C. 7 minutes, (96° C. for 2 minutes, 70° C. for 1 minute, 72° C. for 7 minutes) x30 cycles, 72° C. for 10 minutes).

[0086] The second PCR product was subjected to A-tail addition (the second PCR product 100 ng, 1xExTaq buffer, 2 mM dATP, TAKARA ExTaq; 70° C., 30 minutes), and was cloned to pCR2.1-TOPO vector (Clonetech Co.). As a result, a plasmid for inserting a full-length SsMaT1 was obtained (pCR2.1-SsMat1). Using a DNA sequencer, it was confirmed that an incorrect nucleotide was taken into the DNA sequence of the SsMaT1 gene due to the PCR operation.

[0087] The pCR2.1-SsMaT1 was completely digested with BamHI, and a produced DNA fragment of about 1400 bp was recovered. This DNA fragment was subcloned to the BamHI site of Escherichia coli expression vector pQE-30 (QIAGEN Co.), and was denoted by pQE-30Xa-SsMaT1.

[0088] Expression of SsMaT1 in Escherichia coli containing pQE-30Xa-SsMaT1 was conducted in accordance with the method described in Example 7. Measurement of enzyme activity was conducted in accordance with the method described in Example 2.

[0089] Extract from Escherichia coli expressing the SsMaT1 gene was used to measure enzyme activity, and it was confirmed that malonyl-shisonin was produced in addition to shisonin. Thus, it was confirmed that the SsMaT1 gene encodes a protein having activity for transferring a malonyl group to a glycosyl group at 5-position of flavonoids.

[0090] When acetyl CoA, methylmalonyl CoA, or succinyl CoA was used in place of malonyl CoA as a substrate, a new peak in addition to shisonin was observed in column chromatography using HPLC, indicating that SsMaT1 has activity for transferring these substrates to shisonin.

Example 12 Expression of P. frutescens MaT1

[0091] As in Example 7, using a plasmid constructed such that the PfMaT1 gene obtained in Example 9 can be expressed as a fusion protein fused with LacZ protein, the PfMaT1 gene was expressed in Escherichia coli as in Example 7, and enzyme activity was measured. Measurement of enzyme activity was conducted in accordance with the method described in Example 2.

[0092] An extract from Escherichia coli expressing the PfMaT1 gene was used to measure enzyme activity, and it was confirmed that malonyl-shisonin was produced in addition to shisonin. Thus, it was confirmed that the PfMaT1 gene encodes a protein having activity for transferring a malonyl group to a glycosyl group at 5-position of flavonoids.

INDUSTRIAL APPLICABILITY

[0093] It has been made evident for the first time by the present invention that an aliphatic acyl transferase is involved in the control of flower colours. Flower colour can be altered by expressing this protein in flower petals for modifying anthocyanins. As an aliphatic acyl transferase, any gene which encodes a protein having the same enzyme activity in other organisms may be used in addition to the above-described genes derived from salvia, perilla plant, and lavender.

1 32 1 1419 DNA Salvia guaranitica CDS (3)..(1337) 1 ca aca aca aca ctc ctc gaa aca tgc cac att ccg ccg ccg ccg ccg 47 Thr Thr Thr Leu Leu Glu Thr Cys His Ile Pro Pro Pro Pro Pro 1 5 10 15 gcc aac gac ctc tca atc ccc ctc tcc ttc ttc gac atc aaa tgg ctc 95 Ala Asn Asp Leu Ser Ile Pro Leu Ser Phe Phe Asp Ile Lys Trp Leu 20 25 30 cac tac cac ccc gtc cgc cgc ctc ctc ttc tac cac cac cct tcc tcc 143 His Tyr His Pro Val Arg Arg Leu Leu Phe Tyr His His Pro Ser Ser 35 40 45 aaa tcc caa ttc ctc cac aca atc gtc cca cac ctc aaa caa tca ctc 191 Lys Ser Gln Phe Leu His Thr Ile Val Pro His Leu Lys Gln Ser Leu 50 55 60 tct ctc gct ctc aca cac tac ttc ccc gtc gcc ggc aac ctc ctc tac 239 Ser Leu Ala Leu Thr His Tyr Phe Pro Val Ala Gly Asn Leu Leu Tyr 65 70 75 ccg tcc aac ccc gaa aaa ttc ccc caa ctc cgc tat gcc gcc ggg gat 287 Pro Ser Asn Pro Glu Lys Phe Pro Gln Leu Arg Tyr Ala Ala Gly Asp 80 85 90 95 tcc gtc ccg gtg acg atc gcg gag tcc aat tcc gac ttc gaa agc ctc 335 Ser Val Pro Val Thr Ile Ala Glu Ser Asn Ser Asp Phe Glu Ser Leu 100 105 110 acc gga aac cac acg cgc gac gcc gat caa ttc tac gat ctc ctc ccg 383 Thr Gly Asn His Thr Arg Asp Ala Asp Gln Phe Tyr Asp Leu Leu Pro 115 120 125 cct att cct ccg att gag gag gaa tcg gat tgg aaa ttg atc aac att 431 Pro Ile Pro Pro Ile Glu Glu Glu Ser Asp Trp Lys Leu Ile Asn Ile 130 135 140 ttc gcg gtt cag atc act cta ttc ccc ggc gaa gga atc tgt gtc ggt 479 Phe Ala Val Gln Ile Thr Leu Phe Pro Gly Glu Gly Ile Cys Val Gly 145 150 155 ttc tcc aat cac cac tgc ctc ggc gac gcc aga tct atc gtc gga ttc 527 Phe Ser Asn His His Cys Leu Gly Asp Ala Arg Ser Ile Val Gly Phe 160 165 170 175 atc tcc gct tgg ggt gaa atc aac gga atc gga gga tat gaa gga ttc 575 Ile Ser Ala Trp Gly Glu Ile Asn Gly Ile Gly Gly Tyr Glu Gly Phe 180 185 190 tta tcc aat cgc agt gat tct ctc tcc ctt ccg att ttc gat cga tcg 623 Leu Ser Asn Arg Ser Asp Ser Leu Ser Leu Pro Ile Phe Asp Arg Ser 195 200 205 ttt att aac gat ccg aac aaa atc gac gct att ttc tgg aaa gtg atg 671 Phe Ile Asn Asp Pro Asn Lys Ile Asp Ala Ile Phe Trp Lys Val Met 210 215 220 aga aac ata cct ttg aaa acg gcg tcg ttt ccg ctg cct acg aac aga 719 Arg Asn Ile Pro Leu Lys Thr Ala Ser Phe Pro Leu Pro Thr Asn Arg 225 230 235 gtc aga tct aca ttc ctc ctc cgc aga tcc gac atc gag aag ctg aaa 767 Val Arg Ser Thr Phe Leu Leu Arg Arg Ser Asp Ile Glu Lys Leu Lys 240 245 250 255 acc gcc acc aaa tcg ccg gcg tcg tcg ttc gtc gct gca gca gcg ttc 815 Thr Ala Thr Lys Ser Pro Ala Ser Ser Phe Val Ala Ala Ala Ala Phe 260 265 270 gtc tgg agc tgt atg gtg aaa tcc ggc gac aaa tcc aac gaa aat gcg 863 Val Trp Ser Cys Met Val Lys Ser Gly Asp Lys Ser Asn Glu Asn Ala 275 280 285 ccg gag ctt ttc atc ata cct gcg gac gcc agg ggg agg att gat ccg 911 Pro Glu Leu Phe Ile Ile Pro Ala Asp Ala Arg Gly Arg Ile Asp Pro 290 295 300 ccg ata ccg gag aat tac ttc ggc aac tgc atc gtg agc tcg gtg gcg 959 Pro Ile Pro Glu Asn Tyr Phe Gly Asn Cys Ile Val Ser Ser Val Ala 305 310 315 cgg gtg gag cgc ggg aag ctg ctg gcg gag gac gga ttc gcg gcg gcg 1007 Arg Val Glu Arg Gly Lys Leu Leu Ala Glu Asp Gly Phe Ala Ala Ala 320 325 330 335 gct gaa gca att agc ggg gag atc gag ggg aaa ttg aaa aac aga gat 1055 Ala Glu Ala Ile Ser Gly Glu Ile Glu Gly Lys Leu Lys Asn Arg Asp 340 345 350 gag att ttg aga gga gcg gag aat tgg atg tcg gac ata ttc aaa tgc 1103 Glu Ile Leu Arg Gly Ala Glu Asn Trp Met Ser Asp Ile Phe Lys Cys 355 360 365 ttc ggg atg agc gtg ctc gga gtt tct gga tcg ccg aaa ttc gat ctg 1151 Phe Gly Met Ser Val Leu Gly Val Ser Gly Ser Pro Lys Phe Asp Leu 370 375 380 ttg aag gcg gat ttt gga tgg gga aag gcg agg aaa ttg gag gtg ctg 1199 Leu Lys Ala Asp Phe Gly Trp Gly Lys Ala Arg Lys Leu Glu Val Leu 385 390 395 tcg att gat gga gag aat cac tca atg tcg ctg tgt agc tcg agc gat 1247 Ser Ile Asp Gly Glu Asn His Ser Met Ser Leu Cys Ser Ser Ser Asp 400 405 410 415 ttc aat ggc gga ttg gag gtg ggt ttg tca ttg cct aga gag aga atg 1295 Phe Asn Gly Gly Leu Glu Val Gly Leu Ser Leu Pro Arg Glu Arg Met 420 425 430 gcg gca ttt gca gag gtg ttt act gat gga ctt gcc aat ctt 1337 Ala Ala Phe Ala Glu Val Phe Thr Asp Gly Leu Ala Asn Leu 435 440 445 tgaataattt tcattttata gttattaatt aaatatctta catccaatag taatatctta 1397 attatattcg atattccttc at 1419 2 445 PRT Salvia guaranitica 2 Thr Thr Thr Leu Leu Glu Thr Cys His Ile Pro Pro Pro Pro Pro Ala 1 5 10 15 Asn Asp Leu Ser Ile Pro Leu Ser Phe Phe Asp Ile Lys Trp Leu His 20 25 30 Tyr His Pro Val Arg Arg Leu Leu Phe Tyr His His Pro Ser Ser Lys 35 40 45 Ser Gln Phe Leu His Thr Ile Val Pro His Leu Lys Gln Ser Leu Ser 50 55 60 Leu Ala Leu Thr His Tyr Phe Pro Val Ala Gly Asn Leu Leu Tyr Pro 65 70 75 80 Ser Asn Pro Glu Lys Phe Pro Gln Leu Arg Tyr Ala Ala Gly Asp Ser 85 90 95 Val Pro Val Thr Ile Ala Glu Ser Asn Ser Asp Phe Glu Ser Leu Thr 100 105 110 Gly Asn His Thr Arg Asp Ala Asp Gln Phe Tyr Asp Leu Leu Pro Pro 115 120 125 Ile Pro Pro Ile Glu Glu Glu Ser Asp Trp Lys Leu Ile Asn Ile Phe 130 135 140 Ala Val Gln Ile Thr Leu Phe Pro Gly Glu Gly Ile Cys Val Gly Phe 145 150 155 160 Ser Asn His His Cys Leu Gly Asp Ala Arg Ser Ile Val Gly Phe Ile 165 170 175 Ser Ala Trp Gly Glu Ile Asn Gly Ile Gly Gly Tyr Glu Gly Phe Leu 180 185 190 Ser Asn Arg Ser Asp Ser Leu Ser Leu Pro Ile Phe Asp Arg Ser Phe 195 200 205 Ile Asn Asp Pro Asn Lys Ile Asp Ala Ile Phe Trp Lys Val Met Arg 210 215 220 Asn Ile Pro Leu Lys Thr Ala Ser Phe Pro Leu Pro Thr Asn Arg Val 225 230 235 240 Arg Ser Thr Phe Leu Leu Arg Arg Ser Asp Ile Glu Lys Leu Lys Thr 245 250 255 Ala Thr Lys Ser Pro Ala Ser Ser Phe Val Ala Ala Ala Ala Phe Val 260 265 270 Trp Ser Cys Met Val Lys Ser Gly Asp Lys Ser Asn Glu Asn Ala Pro 275 280 285 Glu Leu Phe Ile Ile Pro Ala Asp Ala Arg Gly Arg Ile Asp Pro Pro 290 295 300 Ile Pro Glu Asn Tyr Phe Gly Asn Cys Ile Val Ser Ser Val Ala Arg 305 310 315 320 Val Glu Arg Gly Lys Leu Leu Ala Glu Asp Gly Phe Ala Ala Ala Ala 325 330 335 Glu Ala Ile Ser Gly Glu Ile Glu Gly Lys Leu Lys Asn Arg Asp Glu 340 345 350 Ile Leu Arg Gly Ala Glu Asn Trp Met Ser Asp Ile Phe Lys Cys Phe 355 360 365 Gly Met Ser Val Leu Gly Val Ser Gly Ser Pro Lys Phe Asp Leu Leu 370 375 380 Lys Ala Asp Phe Gly Trp Gly Lys Ala Arg Lys Leu Glu Val Leu Ser 385 390 395 400 Ile Asp Gly Glu Asn His Ser Met Ser Leu Cys Ser Ser Ser Asp Phe 405 410 415 Asn Gly Gly Leu Glu Val Gly Leu Ser Leu Pro Arg Glu Arg Met Ala 420 425 430 Ala Phe Ala Glu Val Phe Thr Asp Gly Leu Ala Asn Leu 435 440 445 3 1471 DNA Salvia guaranitica CDS (3)..(1322) 3 ca aca aca ctc ctc gaa aca tgc cac att ccg ccg ccg ccg ccg gcc 47 Thr Thr Leu Leu Glu Thr Cys His Ile Pro Pro Pro Pro Pro Ala 1 5 10 15 aac gac ctc tca atc ccc ctc tcc ttc ttc gac atc aaa tgg ctc cac 95 Asn Asp Leu Ser Ile Pro Leu Ser Phe Phe Asp Ile Lys Trp Leu His 20 25 30 tac cac ccc gtc cgc cgc ctc ctc ttc tac cac cac cct tcc tcc aaa 143 Tyr His Pro Val Arg Arg Leu Leu Phe Tyr His His Pro Ser Ser Lys 35 40 45 tcc caa ttc ctc cac aca atc gtt cca cac ctc aaa caa tca ctc tct 191 Ser Gln Phe Leu His Thr Ile Val Pro His Leu Lys Gln Ser Leu Ser 50 55 60 ctc gct ctc aca cac tac ctc ccc gtc gcc ggc aac ctc ctc tac ccg 239 Leu Ala Leu Thr His Tyr Leu Pro Val Ala Gly Asn Leu Leu Tyr Pro 65 70 75 tcc aac ccc gaa aaa ttt ccc caa ctc cgc tat gcc gcc agg gat tcc 287 Ser Asn Pro Glu Lys Phe Pro Gln Leu Arg Tyr Ala Ala Arg Asp Ser 80 85 90 95 gtc ccg gtg acg atc gcg gag tcc aat tcc gac ttc gaa agc ctc acc 335 Val Pro Val Thr Ile Ala Glu Ser Asn Ser Asp Phe Glu Ser Leu Thr 100 105 110 gga aac cac acg cgc gac gcc gat caa ttc tac gat ctc ctc ccg cct 383 Gly Asn His Thr Arg Asp Ala Asp Gln Phe Tyr Asp Leu Leu Pro Pro 115 120 125 att cct ccg att gag gag gaa tcg gat tgg aaa ttg atc aac att ttc 431 Ile Pro Pro Ile Glu Glu Glu Ser Asp Trp Lys Leu Ile Asn Ile Phe 130 135 140 gcg gtt cag atc act cta ttc ccc ggc gaa gga atc tgc gtc ggt ttc 479 Ala Val Gln Ile Thr Leu Phe Pro Gly Glu Gly Ile Cys Val Gly Phe 145 150 155 tcc aat cac cac tgc ctc ggc gac gcc aga tct atg gtc gga ttc atc 527 Ser Asn His His Cys Leu Gly Asp Ala Arg Ser Met Val Gly Phe Ile 160 165 170 175 tcc gct tgg ggt gaa atc aac gga atc gga gga tat gaa gga ttc tta 575 Ser Ala Trp Gly Glu Ile Asn Gly Ile Gly Gly Tyr Glu Gly Phe Leu 180 185 190 tcc aat cac agt gat tct ctc tcc ctt ccg att ttc gat cga tcg ttt 623 Ser Asn His Ser Asp Ser Leu Ser Leu Pro Ile Phe Asp Arg Ser Phe 195 200 205 att aac gat ccg aac aaa atc gac gct att ttc tgg aaa gtg atg aga 671 Ile Asn Asp Pro Asn Lys Ile Asp Ala Ile Phe Trp Lys Val Met Arg 210 215 220 aac ata cct ttg aaa acg gcg tcg ttt ccg ctg cct acg aac aga gtc 719 Asn Ile Pro Leu Lys Thr Ala Ser Phe Pro Leu Pro Thr Asn Arg Val 225 230 235 aga tct aca ttc ctc ctc cgc aga tcc gac atc gag aag ctg aaa acc 767 Arg Ser Thr Phe Leu Leu Arg Arg Ser Asp Ile Glu Lys Leu Lys Thr 240 245 250 255 gcc acc aaa tcg ccg gcg tcg tcg ttc gtc gcg gca gca gcg ttc gtc 815 Ala Thr Lys Ser Pro Ala Ser Ser Phe Val Ala Ala Ala Ala Phe Val 260 265 270 tgg agc tgt atg gtg aaa tcc ggc gac aaa tcc gac gaa aat gcg ccg 863 Trp Ser Cys Met Val Lys Ser Gly Asp Lys Ser Asp Glu Asn Ala Pro 275 280 285 gag ctt ttc atc ata cct gcg gac gcc agg ggg agg att gat ccg ccg 911 Glu Leu Phe Ile Ile Pro Ala Asp Ala Arg Gly Arg Ile Asp Pro Pro 290 295 300 ata ccg gag aat tac ttc ggc aac tgc atc gtg agc tcg gtg gcg cgg 959 Ile Pro Glu Asn Tyr Phe Gly Asn Cys Ile Val Ser Ser Val Ala Arg 305 310 315 gtg gag cgc ggg aag ctg ctg gcg gag gac gga ttc gcg gcg gcg gct 1007 Val Glu Arg Gly Lys Leu Leu Ala Glu Asp Gly Phe Ala Ala Ala Ala 320 325 330 335 gaa gca att ggc ggg gag atc gag ggg aaa ttg aaa aac aga gat gag 1055 Glu Ala Ile Gly Gly Glu Ile Glu Gly Lys Leu Lys Asn Arg Asp Glu 340 345 350 att ttg aga gga gcg gag aat tgg atg tcg gac ata ttc aaa tgc ttc 1103 Ile Leu Arg Gly Ala Glu Asn Trp Met Ser Asp Ile Phe Lys Cys Phe 355 360 365 ggg atg agc gtg ctc gga gtt tct gga tcg ccg aaa ttc gat ctg ttg 1151 Gly Met Ser Val Leu Gly Val Ser Gly Ser Pro Lys Phe Asp Leu Leu 370 375 380 aag gca gat ttc gga tgg gga aag gcg agg aaa ttg gag gtg ctg tcg 1199 Lys Ala Asp Phe Gly Trp Gly Lys Ala Arg Lys Leu Glu Val Leu Ser 385 390 395 att gat gga gag aat cac tca atg tcg ctg tgt agc tcg agc gat ttc 1247 Ile Asp Gly Glu Asn His Ser Met Ser Leu Cys Ser Ser Ser Asp Phe 400 405 410 415 aat ggc gga ttg gag gtg ggt ttg tca ttg cct aga gag aga atg gcg 1295 Asn Gly Gly Leu Glu Val Gly Leu Ser Leu Pro Arg Glu Arg Met Ala 420 425 430 gca ttt gca gag gtg ttt act gat gga ctt gcc aat ctt tgaataatta 1344 Ala Phe Ala Glu Val Phe Thr Asp Gly Leu Ala Asn Leu 435 440 tcattttata gttattaatt aaatatcttg catcccgtcc aatagtaata tcttaattat 1404 attcgatatt ccttcataaa aatattgaca tttgaaataa taacaatcaa attaattaaa 1464 taaaagc 1471 4 444 PRT Artificial Sequence Description of Artificial SequenceAmino acid sequence of malonyltransferase SgMaT1′ of Salvia guaranitica 4 Thr Thr Leu Leu Glu Thr Cys His Ile Pro Pro Pro Pro Pro Ala Asn 1 5 10 15 Asp Leu Ser Ile Pro Leu Ser Phe Phe Asp Ile Lys Trp Leu His Tyr 20 25 30 His Pro Val Arg Arg Leu Leu Phe Tyr His His Pro Ser Ser Lys Ser 35 40 45 Gln Phe Leu His Thr Ile Val Pro His Leu Lys Gln Ser Leu Ser Leu 50 55 60 Ala Leu Thr His Tyr Leu Pro Val Ala Gly Asn Leu Leu Tyr Pro Ser 65 70 75 80 Asn Pro Glu Lys Phe Pro Gln Leu Arg Tyr Ala Ala Arg Asp Ser Val 85 90 95 Pro Val Thr Ile Ala Glu Ser Asn Ser Asp Phe Glu Ser Leu Thr Gly 100 105 110 Asn His Thr Arg Asp Ala Asp Gln Phe Tyr Asp Leu Leu Pro Pro Ile 115 120 125 Pro Pro Ile Glu Glu Glu Ser Asp Trp Lys Leu Ile Asn Ile Phe Ala 130 135 140 Val Gln Ile Thr Leu Phe Pro Gly Glu Gly Ile Cys Val Gly Phe Ser 145 150 155 160 Asn His His Cys Leu Gly Asp Ala Arg Ser Met Val Gly Phe Ile Ser 165 170 175 Ala Trp Gly Glu Ile Asn Gly Ile Gly Gly Tyr Glu Gly Phe Leu Ser 180 185 190 Asn His Ser Asp Ser Leu Ser Leu Pro Ile Phe Asp Arg Ser Phe Ile 195 200 205 Asn Asp Pro Asn Lys Ile Asp Ala Ile Phe Trp Lys Val Met Arg Asn 210 215 220 Ile Pro Leu Lys Thr Ala Ser Phe Pro Leu Pro Thr Asn Arg Val Arg 225 230 235 240 Ser Thr Phe Leu Leu Arg Arg Ser Asp Ile Glu Lys Leu Lys Thr Ala 245 250 255 Thr Lys Ser Pro Ala Ser Ser Phe Val Ala Ala Ala Ala Phe Val Trp 260 265 270 Ser Cys Met Val Lys Ser Gly Asp Lys Ser Asp Glu Asn Ala Pro Glu 275 280 285 Leu Phe Ile Ile Pro Ala Asp Ala Arg Gly Arg Ile Asp Pro Pro Ile 290 295 300 Pro Glu Asn Tyr Phe Gly Asn Cys Ile Val Ser Ser Val Ala Arg Val 305 310 315 320 Glu Arg Gly Lys Leu Leu Ala Glu Asp Gly Phe Ala Ala Ala Ala Glu 325 330 335 Ala Ile Gly Gly Glu Ile Glu Gly Lys Leu Lys Asn Arg Asp Glu Ile 340 345 350 Leu Arg Gly Ala Glu Asn Trp Met Ser Asp Ile Phe Lys Cys Phe Gly 355 360 365 Met Ser Val Leu Gly Val Ser Gly Ser Pro Lys Phe Asp Leu Leu Lys 370 375 380 Ala Asp Phe Gly Trp Gly Lys Ala Arg Lys Leu Glu Val Leu Ser Ile 385 390 395 400 Asp Gly Glu Asn His Ser Met Ser Leu Cys Ser Ser Ser Asp Phe Asn 405 410 415 Gly Gly Leu Glu Val Gly Leu Ser Leu Pro Arg Glu Arg Met Ala Ala 420 425 430 Phe Ala Glu Val Phe Thr Asp Gly Leu Ala Asn Leu 435 440 5 1530 DNA Salvia guaranitica 5 aaaatccaca actttttccc ctccaacctc aaatttccac agccacc atg tcc acc 56 Met Ser Thr 1 acc gtg ctc gaa acc tcc gcc atc tcc cct ccg ccg ggc tcc gcc gcc 104 Thr Val Leu Glu Thr Ser Ala Ile Ser Pro Pro Pro Gly Ser Ala Ala 5 10 15 gac ctc acc ctc ccc ctc tgc ttc ttc gac atc atc tgg ctc cat ttc 152 Asp Leu Thr Leu Pro Leu Cys Phe Phe Asp Ile Ile Trp Leu His Phe 20 25 30 35 cac ccc atc cgc cgc ctc atc ttc tac aac cac cct tgc acc gag gca 200 His Pro Ile Arg Arg Leu Ile Phe Tyr Asn His Pro Cys Thr Glu Ala 40 45 50 gaa ttc tcc tcc acc gtc gtc cca aac ctc aaa cac tcc ctc tct ctc 248 Glu Phe Ser Ser Thr Val Val Pro Asn Leu Lys His Ser Leu Ser Leu 55 60 65 acc ctc caa cac ttc ccc ccc gtc gcc ggc aac ctc ctc ttc cct gtc 296 Thr Leu Gln His Phe Pro Pro Val Ala Gly Asn Leu Leu Phe Pro Val 70 75 80 gac acc gat aaa tcc cgc ccc ttc ctc cgc tac gtc tcc ggc gac acc 344 Asp Thr Asp Lys Ser Arg Pro Phe Leu Arg Tyr Val Ser Gly Asp Thr 85 90 95 gcc ccc ctc act atc gca gtt tcc ggg cgc gac ttc gac gaa tta gtc 392 Ala Pro Leu Thr Ile Ala Val Ser Gly Arg Asp Phe Asp Glu Leu Val 100 105 110 115 gcc gga agc cac gcc cga gac tcc gac caa ttc tac gaa ttc ctc ccc 440 Ala Gly Ser His Ala Arg Asp Ser Asp Gln Phe Tyr Glu Phe Leu Pro 120 125 130 ctg atg ccc ccg atc gcc gag gaa gaa aat tac aaa att gcc cct ctc 488 Leu Met Pro Pro Ile Ala Glu Glu Glu Asn Tyr Lys Ile Ala Pro Leu 135 140 145 atc gcg ctc cag gct acg ctc ttc ccc ggc cgt ggg atc tgc atc ggg 536 Ile Ala Leu Gln Ala Thr Leu Phe Pro Gly Arg Gly Ile Cys Ile Gly 150 155 160 gtg agc aat cac cac tgc ctc ggc gac gcc agg tcg atc gtc gga ttc 584 Val Ser Asn His His Cys Leu Gly Asp Ala Arg Ser Ile Val Gly Phe 165 170 175 gtc tgg gcc tgg gcc gag act aac aga aac aac ggg gac gag cgg ctg 632 Val Trp Ala Trp Ala Glu Thr Asn Arg Asn Asn Gly Asp Glu Arg Leu 180 185 190 195 aga aac cgc tcg ccc ctt ctg att tat gac agg tcg tta gtt ttt ggg 680 Arg Asn Arg Ser Pro Leu Leu Ile Tyr Asp Arg Ser Leu Val Phe Gly 200 205 210 gac acc caa aaa gct gac gaa aag tac tgg agc gtg atg aga aac atc 728 Asp Thr Gln Lys Ala Asp Glu Lys Tyr Trp Ser Val Met Arg Asn Ile 215 220 225 cga cta acg tca tca agt ttt cct gtg cct cgt ggc agg gtc agg gcc 776 Arg Leu Thr Ser Ser Ser Phe Pro Val Pro Arg Gly Arg Val Arg Ala 230 235 240 gcg ttc aca ctg cac cat tca gat att aaa aaa ctc aaa aat aag gtt 824 Ala Phe Thr Leu His His Ser Asp Ile Lys Lys Leu Lys Asn Lys Val 245 250 255 ttg tct aaa aat ccg gac cta gtt ttt gtc tcg tct ttt gca gtc acg 872 Leu Ser Lys Asn Pro Asp Leu Val Phe Val Ser Ser Phe Ala Val Thr 260 265 270 275 gcg gcg tac acg tgg agc tct gtg gtg aag tcc gcg cgc gcg gcc ggg 920 Ala Ala Tyr Thr Trp Ser Ser Val Val Lys Ser Ala Arg Ala Ala Gly 280 285 290 gag gag gtg gat gac gat cga gac gag gtt ttc ttt ttt cct gcg gac 968 Glu Glu Val Asp Asp Asp Arg Asp Glu Val Phe Phe Phe Pro Ala Asp 295 300 305 gcg agg ggc cgg ccg aac gct atg gtt gac cca ccc gtg ccg gtt aat 1016 Ala Arg Gly Arg Pro Asn Ala Met Val Asp Pro Pro Val Pro Val Asn 310 315 320 tac ttc ggg aac tgt tta ggc ggc ggg atg atc aag atg gag cat aag 1064 Tyr Phe Gly Asn Cys Leu Gly Gly Gly Met Ile Lys Met Glu His Lys 325 330 335 aag gtg gcg gcg gag gaa gga ttc gtg gcg gcg gcg gag gcg att gct 1112 Lys Val Ala Ala Glu Glu Gly Phe Val Ala Ala Ala Glu Ala Ile Ala 340 345 350 355 gat caa atc aat aat gtg gtg aat aac aag gag aat ttt ttg aaa gga 1160 Asp Gln Ile Asn Asn Val Val Asn Asn Lys Glu Asn Phe Leu Lys Gly 360 365 370 gcg gat aat tgg ttg tcg gag atg ccg aaa ttt ggg gaa ttg agc act 1208 Ala Asp Asn Trp Leu Ser Glu Met Pro Lys Phe Gly Glu Leu Ser Thr 375 380 385 ttt ggc gtt tcc ggt tcg ccg aaa ttc gat ttg ttg aat tcg gat ttc 1256 Phe Gly Val Ser Gly Ser Pro Lys Phe Asp Leu Leu Asn Ser Asp Phe 390 395 400 ggg tgg ggg acg ggg tcg agg ttg gag gtt ctg tcg atg gat aag gag 1304 Gly Trp Gly Thr Gly Ser Arg Leu Glu Val Leu Ser Met Asp Lys Glu 405 410 415 aag tat tcg atg tcg ttg tgt aat tcg tcg gat tct cct ggc ggt ttg 1352 Lys Tyr Ser Met Ser Leu Cys Asn Ser Ser Asp Ser Pro Gly Gly Leu 420 425 430 435 gtg gtg gga ttg tca ctt cct aag gag agg atg gac gct ttc gca act 1400 Val Val Gly Leu Ser Leu Pro Lys Glu Arg Met Asp Ala Phe Ala Thr 440 445 450 atc ttt gac gat ggt ctt aaa ttt tgagtgtttg attttgttat ttaatttttt 1454 Ile Phe Asp Asp Gly Leu Lys Phe 455 tttaaagttt tgttgcttca aggtataaaa atttaagtca tttgatatga tagattttat 1514 cacgtaagtt tttaac 1530 6 459 PRT Artificial Sequence Description of Artificial SequenceAmino acid sequence of malonyltransferase SgMaT2 of Salvia guaranitica 6 Met Ser Thr Thr Val Leu Glu Thr Ser Ala Ile Ser Pro Pro Pro Gly 1 5 10 15 Ser Ala Ala Asp Leu Thr Leu Pro Leu Cys Phe Phe Asp Ile Ile Trp 20 25 30 Leu His Phe His Pro Ile Arg Arg Leu Ile Phe Tyr Asn His Pro Cys 35 40 45 Thr Glu Ala Glu Phe Ser Ser Thr Val Val Pro Asn Leu Lys His Ser 50 55 60 Leu Ser Leu Thr Leu Gln His Phe Pro Pro Val Ala Gly Asn Leu Leu 65 70 75 80 Phe Pro Val Asp Thr Asp Lys Ser Arg Pro Phe Leu Arg Tyr Val Ser 85 90 95 Gly Asp Thr Ala Pro Leu Thr Ile Ala Val Ser Gly Arg Asp Phe Asp 100 105 110 Glu Leu Val Ala Gly Ser His Ala Arg Asp Ser Asp Gln Phe Tyr Glu 115 120 125 Phe Leu Pro Leu Met Pro Pro Ile Ala Glu Glu Glu Asn Tyr Lys Ile 130 135 140 Ala Pro Leu Ile Ala Leu Gln Ala Thr Leu Phe Pro Gly Arg Gly Ile 145 150 155 160 Cys Ile Gly Val Ser Asn His His Cys Leu Gly Asp Ala Arg Ser Ile 165 170 175 Val Gly Phe Val Trp Ala Trp Ala Glu Thr Asn Arg Asn Asn Gly Asp 180 185 190 Glu Arg Leu Arg Asn Arg Ser Pro Leu Leu Ile Tyr Asp Arg Ser Leu 195 200 205 Val Phe Gly Asp Thr Gln Lys Ala Asp Glu Lys Tyr Trp Ser Val Met 210 215 220 Arg Asn Ile Arg Leu Thr Ser Ser Ser Phe Pro Val Pro Arg Gly Arg 225 230 235 240 Val Arg Ala Ala Phe Thr Leu His His Ser Asp Ile Lys Lys Leu Lys 245 250 255 Asn Lys Val Leu Ser Lys Asn Pro Asp Leu Val Phe Val Ser Ser Phe 260 265 270 Ala Val Thr Ala Ala Tyr Thr Trp Ser Ser Val Val Lys Ser Ala Arg 275 280 285 Ala Ala Gly Glu Glu Val Asp Asp Asp Arg Asp Glu Val Phe Phe Phe 290 295 300 Pro Ala Asp Ala Arg Gly Arg Pro Asn Ala Met Val Asp Pro Pro Val 305 310 315 320 Pro Val Asn Tyr Phe Gly Asn Cys Leu Gly Gly Gly Met Ile Lys Met 325 330 335 Glu His Lys Lys Val Ala Ala Glu Glu Gly Phe Val Ala Ala Ala Glu 340 345 350 Ala Ile Ala Asp Gln Ile Asn Asn Val Val Asn Asn Lys Glu Asn Phe 355 360 365 Leu Lys Gly Ala Asp Asn Trp Leu Ser Glu Met Pro Lys Phe Gly Glu 370 375 380 Leu Ser Thr Phe Gly Val Ser Gly Ser Pro Lys Phe Asp Leu Leu Asn 385 390 395 400 Ser Asp Phe Gly Trp Gly Thr Gly Ser Arg Leu Glu Val Leu Ser Met 405 410 415 Asp Lys Glu Lys Tyr Ser Met Ser Leu Cys Asn Ser Ser Asp Ser Pro 420 425 430 Gly Gly Leu Val Val Gly Leu Ser Leu Pro Lys Glu Arg Met Asp Ala 435 440 445 Phe Ala Thr Ile Phe Asp Asp Gly Leu Lys Phe 450 455 7 15 PRT Artificial Sequence Descrition of Artificial SequencePartial amino acid sequence of malonyltransferase 7 Tyr Ala Ala Gly Asp Ser Val Pro Val Thr Ile Ala Ala Ser Asn 1 5 10 15 8 10 PRT Artificial Sequence Descrition of Artificial SequencePartial amino acid sequence of malonyltransferase 8 Leu Leu Phe Tyr His His Pro Ser Ser Lys 1 5 10 9 19 PRT Artificial Sequence Description of Artificial SequencePartial amino acid sequence of malonyltransferase 9 Ser Gly Asp Lys Ser Asp Glu Asn Ala Pro Glu Leu Phe Ile Ile Pro 1 5 10 15 Ala Asp Ala 10 8 PRT Artificial Sequence Description of Artificial SequencePartial amino acid sequence of malonyltransferase 10 Met Ala Ala Phe Glu Glu Val Phe 1 5 11 7 PRT Artificial Sequence Description of Artificial SequencePartial amino acid sequence of malonyltransferase 11 Trp Leu His Tyr His Pro Val 1 5 12 11 PRT Artificial Sequence Description of Artificial SequencePartial amino acid sequence of malonyltransferase 12 Gly Ala Glu Asn Trp Met Ser Asp Ile Phe Lys 1 5 10 13 20 PRT Artificial Sequence Description of Artificial SequencePartial amino acid sequence of malonyltransferase 13 Leu Ala Ala Glu Xaa Gly Phe Ala Val Ala Ala Ala Ala Ile Gly Gly 1 5 10 15 Gly Ile Ile Gly 20 14 13 PRT Artificial Sequence Description of Artificial SequencePartial amino acid sequence of malonyltransferase 14 Ser Phe Ile Asn Asp Pro Asn Lys Ile Asp Ala Ile Phe 1 5 10 15 10 PRT Artificial Sequence Description of Artificial SequencePartial amino acid sequence of malonyltransferase 15 Thr Ala Ser Phe Pro Leu Pro Thr Asn Arg 1 5 10 16 5 PRT Artificial Sequence Description of Artificial SequencePartial amino acid sequence of malonyltransferase 16 Phe Pro Gln Leu Arg 1 5 17 7 PRT Artificial Sequence Description of Artificial SequencePartial amino acid sequence of malonyltransferase 17 Ala Asp Phe Gly Trp Gly Lys 1 5 18 14 PRT Artificial Sequence Description of Artificial SequencePartial amino acid sequence of malonyltransferase 18 Asp Ala Asp Gln Phe Tyr Asp Leu Leu Pro Pro Ile Pro Pro 1 5 10 19 26 DNA Artificial Sequence Description of Artificial SequencePrimer 19 taygcngcng gngaytcngt nccngt 26 20 20 DNA Artificial Sequence Description of Artificial SequencePrimer 20 gtnccngtna cnathgcngc 20 21 21 DNA Artificial Sequence Description of Artificial SequencePrimer 21 yttnccccan ccraartcng c 21 22 1636 DNA Salvia splendens 22 ca aca aca aca aca atc ctc gaa aca tgc cac att cca ccg ccg ccg 47 Thr Thr Thr Thr Ile Leu Glu Thr Cys His Ile Pro Pro Pro Pro 1 5 10 15 gcg gcc aac gac ctc tca atc ccc ctc tcc ttc ttc gac atc aaa tgg 95 Ala Ala Asn Asp Leu Ser Ile Pro Leu Ser Phe Phe Asp Ile Lys Trp 20 25 30 ctc cac tac cac ccc gtc cgc cgc ctc ctc ttc tac cac cac cct tcc 143 Leu His Tyr His Pro Val Arg Arg Leu Leu Phe Tyr His His Pro Ser 35 40 45 tcc aaa tcc caa ttc ctc cac aca atc gtt cca cac ctc aaa caa tca 191 Ser Lys Ser Gln Phe Leu His Thr Ile Val Pro His Leu Lys Gln Ser 50 55 60 ctc tct ctc gct ctc aca cac tac ctc ccc gtc gcc ggc aac ctc ctc 239 Leu Ser Leu Ala Leu Thr His Tyr Leu Pro Val Ala Gly Asn Leu Leu 65 70 75 tac ccg tcc aac acc gaa aaa ttc ccc caa ctc cgc tac gcc gcc ggg 287 Tyr Pro Ser Asn Thr Glu Lys Phe Pro Gln Leu Arg Tyr Ala Ala Gly 80 85 90 95 gat tcc gtc ccg gtg acg atc gcg gag tcc aat tcc gac ttc gaa agc 335 Asp Ser Val Pro Val Thr Ile Ala Glu Ser Asn Ser Asp Phe Glu Ser 100 105 110 ctt acc gga aac cac acg cgc gac gcc gat caa ttc tac gat ctc ctc 383 Leu Thr Gly Asn His Thr Arg Asp Ala Asp Gln Phe Tyr Asp Leu Leu 115 120 125 ccg cct att cct ccg att gag gag gaa tcg gat tgg aaa ttg atc aac 431 Pro Pro Ile Pro Pro Ile Glu Glu Glu Ser Asp Trp Lys Leu Ile Asn 130 135 140 att ttc gcg gtt cag atc act ctg ttc ccc ggc gaa gga atc tgc atc 479 Ile Phe Ala Val Gln Ile Thr Leu Phe Pro Gly Glu Gly Ile Cys Ile 145 150 155 ggt ttc tcc aat cac cac tgc ctc ggc gac gcc aga tct atc gtc gga 527 Gly Phe Ser Asn His His Cys Leu Gly Asp Ala Ser Ser Ile Val Gly 160 165 170 175 ttc atc tcc gct tgg ggt gaa atc aac gga atc gga gga tat gaa gga 575 Phe Ile Ser Ala Trp Gly Glu Ile Asn Gly Ile Gly Gly Tyr Glu Gly 180 185 190 ttc tta tcc aat cac agt gat tct ctc tcc ctt ccg att ttc gat cga 623 Phe Leu Ser Asn His Ser Asp Ser Leu Ser Leu Pro Ile Phe Asp Arg 195 200 205 tcg ttt att aac gat ccg aac aaa atc gac gct att ttc tgg aaa gtg 671 Ser Phe Ile Asn Asp Pro Asn Lys Ile Asp Ala Ile Phe Trp Lys Val 210 215 220 ctg aga aac ata cca ttg aaa acg gcg tcg ttt ccg ctg cct acg aac 719 Leu Ser Asn Met Pro Leu Lys Thr Ala Ser Phe Pro Leu Pro Thr Asn 225 230 235 aga gtc aga tct aca ttc ctc ctc cgc aga tcc gac atc gag aag ctg 767 Ser Val Ser Ser Thr Phe Leu Leu Arg Ser Ser Asp Ile Glu Lys Leu 240 245 250 255 aaa acc gcc act aaa tcg ccg gcg tcg tcg ttc gtc gcg gca gca gcg 815 Lys Thr Ala Thr Lys Ser Pro Ala Ser Ser Phe Val Ala Ala Ala Ala 260 265 270 ttc gtc tgg agc tgt atg gtg aaa tcc ggc gac aaa tcc gac gaa aat 863 Phe Val Trp Ser Cys Met Val Lys Ser Gly Asp Lys Ser Asp Glu Asn 275 280 285 gcg cct gag ctt ttc atc ata cct gcg gac gcc agg ggg agg gtg gat 911 Ala Pro Glu Leu Phe Ile Met Pro Ala Asp Ala Ser Gly Ser Val Asp 290 295 300 ccg ccg ata ccg gag aat tac ttc ggc aac tgc atc gtg agc tcg gtg 959 Pro Pro Met Pro Glu Asn Tyr Phe Gly Asn Cys Ile Val Ser Ser Val 305 310 315 gcg cag gtg gag cgc ggg aag ctg gcg gcg gag gat gga ttc gcg gtg 1007 Ala Gln Val Glu Arg Gly Lys Leu Ala Ala Glu Asp Gly Phe Ala Val 320 325 330 335 gcg gct gaa gca att ggc ggg gag atc gag ggg aaa ttg aaa aac aga 1055 Ala Ala Glu Ala Ile Gly Gly Glu Ile Glu Gly Lys Leu Lys Asn Ser 340 345 350 gat gag att ttg aga gga gcg gag aat tgg atg tcg gac ata ttc aaa 1103 Asp Glu Ile Leu Ser Gly Ala Glu Asn Trp Met Ser Asp Met Phe Lys 355 360 365 tgc ttc ggg atg agc gtg ctc gga gtt tct gga tcg ccg aaa ttc gat 1151 Cys Phe Gly Met Ser Val Leu Gly Val Ser Gly Ser Pro Lys Phe Asp 370 375 380 ctg ttg aag gcg gat ttt gga tgg gga aag gcg agg aaa ttg gag gtg 1199 Leu Leu Lys Ala Asp Phe Gly Trp Gly Lys Ala Ser Lys Leu Glu Val 385 390 395 ctg tcg att gat gga gag aat cac tca atg tcg ctg tgt agc tcg agc 1247 Leu Ser Ile Asp Gly Glu Asn His Ser Met Ser Leu Cys Ser Ser Ser 400 405 410 415 gat ttc aat ggc gga ttg gag gtg ggt ttg tcg ttg ccc aga gag aga 1295 Asp Phe Asn Gly Gly Leu Glu Val Gly Leu Ser Leu Pro Ser Glu Ser 420 425 430 atg gcg gca ttt gaa gag gtg ttt aga gca tcc ata atg gcg gcg agc 1343 Met Ala Ala Phe Glu Glu Val Phe Ser Ala Ser Met Met Ala Ala Ser 435 440 445 gga ccg gct agg cga tct ccg gcg ctc gtc gaa cca ttg taaccggcga 1392 Gly Pro Ala Ser Arg Ser Pro Ala Leu Val Glu Pro Leu 450 455 460 gcgccatttc ggcgaaaaaa tcggcgagcg caggccgatt cgcgagagcg ctgggcgatg 1452 cgctcgccgc cattgcaggc tccggaccgg ggagcgatcg gcgagcaaaa ttttcttttt 1512 cttttaattt tcgaaactct atatgtacgc gttttgcacg tcattttcat tcgcatcact 1572 tgttttaacg agtactctct ctatcttaat ttctatataa gatcaacaac gtgaaatgaa 1632 gaac 1636 23 460 PRT Salvia splendens 23 Thr Thr Thr Thr Ile Leu Glu Thr Cys His Ile Pro Pro Pro Pro Ala 1 5 10 15 Ala Asn Asp Leu Ser Ile Pro Leu Ser Phe Phe Asp Ile Lys Trp Leu 20 25 30 His Tyr His Pro Val Arg Arg Leu Leu Phe Tyr His His Pro Ser Ser 35 40 45 Lys Ser Gln Phe Leu His Thr Ile Val Pro His Leu Lys Gln Ser Leu 50 55 60 Ser Leu Ala Leu Thr His Tyr Leu Pro Val Ala Gly Asn Leu Leu Tyr 65 70 75 80 Pro Ser Asn Thr Glu Lys Phe Pro Gln Leu Arg Tyr Ala Ala Gly Asp 85 90 95 Ser Val Pro Val Thr Ile Ala Glu Ser Asn Ser Asp Phe Glu Ser Leu 100 105 110 Thr Gly Asn His Thr Arg Asp Ala Asp Gln Phe Tyr Asp Leu Leu Pro 115 120 125 Pro Ile Pro Pro Ile Glu Glu Glu Ser Asp Trp Lys Leu Ile Asn Ile 130 135 140 Phe Ala Val Gln Ile Thr Leu Phe Pro Gly Glu Gly Ile Cys Ile Gly 145 150 155 160 Phe Ser Asn His His Cys Leu Gly Asp Ala Ser Ser Ile Val Gly Phe 165 170 175 Ile Ser Ala Trp Gly Glu Ile Asn Gly Ile Gly Gly Tyr Glu Gly Phe 180 185 190 Leu Ser Asn His Ser Asp Ser Leu Ser Leu Pro Ile Phe Asp Arg Ser 195 200 205 Phe Ile Asn Asp Pro Asn Lys Ile Asp Ala Ile Phe Trp Lys Val Leu 210 215 220 Ser Asn Met Pro Leu Lys Thr Ala Ser Phe Pro Leu Pro Thr Asn Ser 225 230 235 240 Val Ser Ser Thr Phe Leu Leu Arg Ser Ser Asp Ile Glu Lys Leu Lys 245 250 255 Thr Ala Thr Lys Ser Pro Ala Ser Ser Phe Val Ala Ala Ala Ala Phe 260 265 270 Val Trp Ser Cys Met Val Lys Ser Gly Asp Lys Ser Asp Glu Asn Ala 275 280 285 Pro Glu Leu Phe Ile Met Pro Ala Asp Ala Ser Gly Ser Val Asp Pro 290 295 300 Pro Met Pro Glu Asn Tyr Phe Gly Asn Cys Ile Val Ser Ser Val Ala 305 310 315 320 Gln Val Glu Arg Gly Lys Leu Ala Ala Glu Asp Gly Phe Ala Val Ala 325 330 335 Ala Glu Ala Ile Gly Gly Glu Ile Glu Gly Lys Leu Lys Asn Ser Asp 340 345 350 Glu Ile Leu Ser Gly Ala Glu Asn Trp Met Ser Asp Met Phe Lys Cys 355 360 365 Phe Gly Met Ser Val Leu Gly Val Ser Gly Ser Pro Lys Phe Asp Leu 370 375 380 Leu Lys Ala Asp Phe Gly Trp Gly Lys Ala Ser Lys Leu Glu Val Leu 385 390 395 400 Ser Ile Asp Gly Glu Asn His Ser Met Ser Leu Cys Ser Ser Ser Asp 405 410 415 Phe Asn Gly Gly Leu Glu Val Gly Leu Ser Leu Pro Ser Glu Ser Met 420 425 430 Ala Ala Phe Glu Glu Val Phe Ser Ala Ser Met Met Ala Ala Ser Gly 435 440 445 Pro Ala Ser Arg Ser Pro Ala Leu Val Glu Pro Leu 450 455 460 24 1498 DNA Salvia splendens 24 gcaacttttt cccctccaac ctataatttc cacaaccacc atg acc acc acc 52 Met Thr Thr Thr 1 gtg ctc gaa acc tcc gcc atc tcc cct ccg ccg ggc tcc gcc gcc gac 100 Val Leu Glu Thr Ser Ala Ile Ser Pro Pro Pro Gly Ser Ala Ala Asp 5 10 15 20 ctc acc ctc ccc ctc tgc ttc ttc gac atc atc tgg ctc cat ttc cac 148 Leu Thr Leu Pro Leu Cys Phe Phe Asp Ile Ile Trp Leu His Phe His 25 30 35 ccc atc cgc cgc ctc atc ttc tac aac cac cct tgc acc gag gcc gaa 196 Pro Ile Arg Arg Leu Ile Phe Tyr Asn His Pro Cys Thr Glu Ala Glu 40 45 50 ttc tcc tcc acc atc gtc cca aac ctc aaa cac tcc ctc tct ctc acc 244 Phe Ser Ser Thr Ile Val Pro Asn Leu Lys His Ser Leu Ser Leu Thr 55 60 65 ctc caa cac ttc ccc ccc gtc gcc ggc aac ctc ctc ttc cct gtc gac 292 Leu Gln His Phe Pro Pro Val Ala Gly Asn Leu Leu Phe Pro Val Asp 70 75 80 acc gat aaa tcc cgc ccc ttc ctc cgc tac gtc tcc ggc gac acc gcc 340 Thr Asp Lys Ser Arg Pro Phe Leu Arg Tyr Val Ser Gly Asp Thr Ala 85 90 95 100 ccc ctc acg atc gcc gtc tcc ggg cgc gac ttc gac gaa tta gtc gct 388 Pro Leu Thr Ile Ala Val Ser Gly Arg Asp Phe Asp Glu Leu Val Ala 105 110 115 ggc agc cgc gcc cga gac tcc gac caa ttc tac gaa ttc ctc ccc ctg 436 Gly Ser Arg Ala Arg Asp Ser Asp Gln Phe Tyr Glu Phe Leu Pro Leu 120 125 130 atg ccc ccg atc gcc gag gag gaa gat tac aaa att gcc cct ctc atc 484 Met Pro Pro Ile Ala Glu Glu Glu Asp Tyr Lys Ile Ala Pro Leu Ile 135 140 145 gcg ctc cag gcc acg ctc ttc ccc ggc cgc ggg atc tgc atc ggg gtg 532 Ala Leu Gln Ala Thr Leu Phe Pro Gly Arg Gly Ile Cys Ile Gly Val 150 155 160 agc aat cac cac tgc ctc ggt gac gcc agg tcg atc gtt gca ttc gtc 580 Ser Asn His His Cys Leu Gly Asp Ala Arg Ser Ile Val Ala Phe Val 165 170 175 180 tcg gcc tgg gcc gag acg aac aga aac agc ggg gac gag cgg ctg aga 628 Ser Ala Trp Ala Glu Thr Asn Arg Asn Ser Gly Asp Glu Arg Leu Arg 185 190 195 aac tgc acg ctc ccg ctg att tat gat agg tcg tca gtt ttt ggg gac 676 Asn Cys Thr Leu Pro Leu Ile Tyr Asp Arg Ser Ser Val Phe Gly Asp 200 205 210 acc caa aaa gct gac gaa aag tac tgg agc gtg atg aga aac atc ccg 724 Thr Gln Lys Ala Asp Glu Lys Tyr Trp Ser Val Met Arg Asn Ile Pro 215 220 225 ctg aca tca tca agt ttt cct gtg cct agt ggc agg gtc agg gcc gcg 772 Leu Thr Ser Ser Ser Phe Pro Val Pro Ser Gly Arg Val Arg Ala Ala 230 235 240 ttc aca ctg cac cag tca gat att aaa aac ctc aaa aat aag gtt ttg 820 Phe Thr Leu His Gln Ser Asp Ile Lys Asn Leu Lys Asn Lys Val Leu 245 250 255 260 tct aaa aat ccg gac cta gtt ttc gtc tcg tct ttt gcc gtc acg gcg 868 Ser Lys Asn Pro Asp Leu Val Phe Val Ser Ser Phe Ala Val Thr Ala 265 270 275 gcg tac acg tgg agc tct gtg gtg aag tcc gcg cgc gcg gcc ggg gag 916 Ala Tyr Thr Trp Ser Ser Val Val Lys Ser Ala Arg Ala Ala Gly Glu 280 285 290 gag gtg gat gac gat cgt gac gag gtt ttc ttt ttt cct gcg gac gcg 964 Glu Val Asp Asp Asp Arg Asp Glu Val Phe Phe Phe Pro Ala Asp Ala 295 300 305 agg ggt cgg ccg aac gct atg gtt gac ccg ccc gtg ccg gtt aat tac 1012 Arg Gly Arg Pro Asn Ala Met Val Asp Pro Pro Val Pro Val Asn Tyr 310 315 320 ttc ggg aac tgt tta ggc ggc ggg atg atc aag atg gag cat aag aag 1060 Phe Gly Asn Cys Leu Gly Gly Gly Met Ile Lys Met Glu His Lys Lys 325 330 335 340 gtg gcg gcg gag gaa gga ttc gtg gcg gcg gcg gag gcg att gct gat 1108 Val Ala Ala Glu Glu Gly Phe Val Ala Ala Ala Glu Ala Ile Ala Asp 345 350 355 caa atc aat aat gtg gtg aat aac aag gat aat ttt ttg aaa gga gcg 1156 Gln Ile Asn Asn Val Val Asn Asn Lys Asp Asn Phe Leu Lys Gly Ala 360 365 370 gat aat tgg ttg tcg gag atg ccg aaa ttt ggg gaa ttg agc act ttt 1204 Asp Asn Trp Leu Ser Glu Met Pro Lys Phe Gly Glu Leu Ser Thr Phe 375 380 385 ggc gtt tcc ggt tcg ccg aaa ttc gat ttg ttg aat tcg gat ttc ggg 1252 Gly Val Ser Gly Ser Pro Lys Phe Asp Leu Leu Asn Ser Asp Phe Gly 390 395 400 tgg ggg acg ggg tcg agg ttg gag gtt ctg tcg atg gat aag gag aag 1300 Trp Gly Thr Gly Ser Arg Leu Glu Val Leu Ser Met Asp Lys Glu Lys 405 410 415 420 tat tcg atg tcg ttg tgt aat tcg tcg gat tct cct ggc ggt ttg gtg 1348 Tyr Ser Met Ser Leu Cys Asn Ser Ser Asp Ser Pro Gly Gly Leu Val 425 430 435 gtc gga ttg tca ctt cct aag gag agg atg gat gct ttc gca act atc 1396 Val Gly Leu Ser Leu Pro Lys Glu Arg Met Asp Ala Phe Ala Thr Ile 440 445 450 ttt gaa gat ggt ctt aaa ttt tgagtgtttg attttgttat ttaatttttt 1447 Phe Glu Asp Gly Leu Lys Phe 455 tttaaagttt tgttgcttca agggttaaaa atttaagtca tttgatatga t 1498 25 459 PRT Salvia splendens 25 Met Thr Thr Thr Val Leu Glu Thr Ser Ala Ile Ser Pro Pro Pro Gly 1 5 10 15 Ser Ala Ala Asp Leu Thr Leu Pro Leu Cys Phe Phe Asp Ile Ile Trp 20 25 30 Leu His Phe His Pro Ile Arg Arg Leu Ile Phe Tyr Asn His Pro Cys 35 40 45 Thr Glu Ala Glu Phe Ser Ser Thr Ile Val Pro Asn Leu Lys His Ser 50 55 60 Leu Ser Leu Thr Leu Gln His Phe Pro Pro Val Ala Gly Asn Leu Leu 65 70 75 80 Phe Pro Val Asp Thr Asp Lys Ser Arg Pro Phe Leu Arg Tyr Val Ser 85 90 95 Gly Asp Thr Ala Pro Leu Thr Ile Ala Val Ser Gly Arg Asp Phe Asp 100 105 110 Glu Leu Val Ala Gly Ser Arg Ala Arg Asp Ser Asp Gln Phe Tyr Glu 115 120 125 Phe Leu Pro Leu Met Pro Pro Ile Ala Glu Glu Glu Asp Tyr Lys Ile 130 135 140 Ala Pro Leu Ile Ala Leu Gln Ala Thr Leu Phe Pro Gly Arg Gly Ile 145 150 155 160 Cys Ile Gly Val Ser Asn His His Cys Leu Gly Asp Ala Arg Ser Ile 165 170 175 Val Ala Phe Val Ser Ala Trp Ala Glu Thr Asn Arg Asn Ser Gly Asp 180 185 190 Glu Arg Leu Arg Asn Cys Thr Leu Pro Leu Ile Tyr Asp Arg Ser Ser 195 200 205 Val Phe Gly Asp Thr Gln Lys Ala Asp Glu Lys Tyr Trp Ser Val Met 210 215 220 Arg Asn Ile Pro Leu Thr Ser Ser Ser Phe Pro Val Pro Ser Gly Arg 225 230 235 240 Val Arg Ala Ala Phe Thr Leu His Gln Ser Asp Ile Lys Asn Leu Lys 245 250 255 Asn Lys Val Leu Ser Lys Asn Pro Asp Leu Val Phe Val Ser Ser Phe 260 265 270 Ala Val Thr Ala Ala Tyr Thr Trp Ser Ser Val Val Lys Ser Ala Arg 275 280 285 Ala Ala Gly Glu Glu Val Asp Asp Asp Arg Asp Glu Val Phe Phe Phe 290 295 300 Pro Ala Asp Ala Arg Gly Arg Pro Asn Ala Met Val Asp Pro Pro Val 305 310 315 320 Pro Val Asn Tyr Phe Gly Asn Cys Leu Gly Gly Gly Met Ile Lys Met 325 330 335 Glu His Lys Lys Val Ala Ala Glu Glu Gly Phe Val Ala Ala Ala Glu 340 345 350 Ala Ile Ala Asp Gln Ile Asn Asn Val Val Asn Asn Lys Asp Asn Phe 355 360 365 Leu Lys Gly Ala Asp Asn Trp Leu Ser Glu Met Pro Lys Phe Gly Glu 370 375 380 Leu Ser Thr Phe Gly Val Ser Gly Ser Pro Lys Phe Asp Leu Leu Asn 385 390 395 400 Ser Asp Phe Gly Trp Gly Thr Gly Ser Arg Leu Glu Val Leu Ser Met 405 410 415 Asp Lys Glu Lys Tyr Ser Met Ser Leu Cys Asn Ser Ser Asp Ser Pro 420 425 430 Gly Gly Leu Val Val Gly Leu Ser Leu Pro Lys Glu Arg Met Asp Ala 435 440 445 Phe Ala Thr Ile Phe Glu Asp Gly Leu Lys Phe 450 455 26 1502 DNA Perilla frutescens 26 aattaacata tatttatatt tagtcc atg aca aca aca ttg ctc gaa 47 Met Thr Thr Thr Leu Leu Glu 1 5 acc tgc cgg att ctg cca ccg ccg acc gac gag gtc tcg atc cct ctc 95 Thr Cys Arg Ile Leu Pro Pro Pro Thr Asp Glu Val Ser Ile Pro Leu 10 15 20 tct ttc ttc gac atg aag tgg ctc cac ttc cac ccc ctc cgc cgt ctc 143 Ser Phe Phe Asp Met Lys Trp Leu His Phe His Pro Leu Arg Arg Leu 25 30 35 ctc ttc tac gac cac cct tgt tcc aag ccc caa ttc ttg gat gcc att 191 Leu Phe Tyr Asp His Pro Cys Ser Lys Pro Gln Phe Leu Asp Ala Ile 40 45 50 55 gtt cca cac ctc aaa caa tct ctc tcc ctc act ctc aaa cac tac ctc 239 Val Pro His Leu Lys Gln Ser Leu Ser Leu Thr Leu Lys His Tyr Leu 60 65 70 ccc gtc gcc ggc aat ctg ctc tac cct tca tca aac acc gac caa aag 287 Pro Val Ala Gly Asn Leu Leu Tyr Pro Ser Ser Asn Thr Asp Gln Lys 75 80 85 ccc cga ctt cgc tgc gtc gcc ggg gat tca gtc ccg ctg acg atc gcg 335 Pro Arg Leu Arg Cys Val Ala Gly Asp Ser Val Pro Leu Thr Ile Ala 90 95 100 gag tcc acc acc gat ttc gac atg ctc acc gga aat cat gca aga gat 383 Glu Ser Thr Thr Asp Phe Asp Met Leu Thr Gly Asn His Ala Arg Asp 105 110 115 gcc gat cag ttc tac gat ttc gtg gcg ccg atg cca cct att gca gag 431 Ala Asp Gln Phe Tyr Asp Phe Val Ala Pro Met Pro Pro Ile Ala Glu 120 125 130 135 gaa ttc gaa tgc aaa ata gtt ccc gtt ttc tcc ctg caa gtg acg ctg 479 Glu Phe Glu Cys Lys Ile Val Pro Val Phe Ser Leu Gln Val Thr Leu 140 145 150 ttt cct ggg cgt gga att tgc atc ggt tta tcc aat cat cac tgc ctc 527 Phe Pro Gly Arg Gly Ile Cys Ile Gly Leu Ser Asn His His Cys Leu 155 160 165 ggc gac gcc aga tcg gtg gtg gga ttc gtg ttg gcg tgg gct tcc atc 575 Gly Asp Ala Arg Ser Val Val Gly Phe Val Leu Ala Trp Ala Ser Ile 170 175 180 aat aaa ttc ggt ggt gat gag gag ttt ctg tcg gaa aac ggt gaa tct 623 Asn Lys Phe Gly Gly Asp Glu Glu Phe Leu Ser Glu Asn Gly Glu Ser 185 190 195 ttg ccg att ttt gat cga tct ttg att aag gat cca ctc gaa atc gat 671 Leu Pro Ile Phe Asp Arg Ser Leu Ile Lys Asp Pro Leu Glu Ile Asp 200 205 210 215 act att ttc tgg aaa gta ttg aga aac ata cct ctg aag ccg tca tct 719 Thr Ile Phe Trp Lys Val Leu Arg Asn Ile Pro Leu Lys Pro Ser Ser 220 225 230 ttt ccg tta ccc acc aac aga gtc aga gcc aca ttc gtt ctc agt caa 767 Phe Pro Leu Pro Thr Asn Arg Val Arg Ala Thr Phe Val Leu Ser Gln 235 240 245 tcc gac ata aaa agg cta aaa cat ttg gcg aat aac aac cta gtt caa 815 Ser Asp Ile Lys Arg Leu Lys His Leu Ala Asn Asn Asn Leu Val Gln 250 255 260 ccg tcg tct ttc gtc gtc gcg gct gcg tat att tgg agc tgc atg gtg 863 Pro Ser Ser Phe Val Val Ala Ala Ala Tyr Ile Trp Ser Cys Met Val 265 270 275 aaa tcc ggc gac gga ggt gag gct aac gcg ccg gaa ttg ttc gtt att 911 Lys Ser Gly Asp Gly Gly Glu Ala Asn Ala Pro Glu Leu Phe Val Ile 280 285 290 295 cca gcc gac gcg aga ggc cgg acg aat ccg ccg gtg ccg gcg aat tac 959 Pro Ala Asp Ala Arg Gly Arg Thr Asn Pro Pro Val Pro Ala Asn Tyr 300 305 310 ttc ggg aat tgc ata gtt ggc ggg gta gta aaa gtg gag cac gaa aag 1007 Phe Gly Asn Cys Ile Val Gly Gly Val Val Lys Val Glu His Glu Lys 315 320 325 atg gcg gga aac gag gga ttt gtg att gct gca gaa gcc ata gct ggg 1055 Met Ala Gly Asn Glu Gly Phe Val Ile Ala Ala Glu Ala Ile Ala Gly 330 335 340 gaa atc aag aac aag atg aat gat aaa gag gag att ttg aaa ggg gcg 1103 Glu Ile Lys Asn Lys Met Asn Asp Lys Glu Glu Ile Leu Lys Gly Ala 345 350 355 gag aat tgg ctg tcg gaa atc tgg aaa tgt atg ggg atg agc gtg ctc 1151 Glu Asn Trp Leu Ser Glu Ile Trp Lys Cys Met Gly Met Ser Val Leu 360 365 370 375 gga att tct ggt tcg ccg aaa ttc gat tta tcg aat gca gat ttt gga 1199 Gly Ile Ser Gly Ser Pro Lys Phe Asp Leu Ser Asn Ala Asp Phe Gly 380 385 390 tgg gga aag gcg agg aaa ctg gaa gtt gtg tcg atc gat gga gag aag 1247 Trp Gly Lys Ala Arg Lys Leu Glu Val Val Ser Ile Asp Gly Glu Lys 395 400 405 tat acg atg tcg ttg tgt aat tcc gac tgt ggg ttg gag gtt ggg ttg 1295 Tyr Thr Met Ser Leu Cys Asn Ser Asp Cys Gly Leu Glu Val Gly Leu 410 415 420 tcg ttg ccg gga gaa aga atg gaa gct ttt gca gcc ata ttt gcc gat 1343 Ser Leu Pro Gly Glu Arg Met Glu Ala Phe Ala Ala Ile Phe Ala Asp 425 430 435 ggc cta gct aag cta gat agc tca tgattcatga ataatatata tatatatata 1397 Gly Leu Ala Lys Leu Asp Ser Ser 440 445 tatagagaga gagagagaat tgatatgccc atctttgtgg gcgccgctga tcgtcatcac 1457 tttatttatt cttttttttt ttggtaattt tcgcttttct cccag 1502 27 447 PRT Perilla frutescens 27 Met Thr Thr Thr Leu Leu Glu Thr Cys Arg Ile Leu Pro Pro Pro Thr 1 5 10 15 Asp Glu Val Ser Ile Pro Leu Ser Phe Phe Asp Met Lys Trp Leu His 20 25 30 Phe His Pro Leu Arg Arg Leu Leu Phe Tyr Asp His Pro Cys Ser Lys 35 40 45 Pro Gln Phe Leu Asp Ala Ile Val Pro His Leu Lys Gln Ser Leu Ser 50 55 60 Leu Thr Leu Lys His Tyr Leu Pro Val Ala Gly Asn Leu Leu Tyr Pro 65 70 75 80 Ser Ser Asn Thr Asp Gln Lys Pro Arg Leu Arg Cys Val Ala Gly Asp 85 90 95 Ser Val Pro Leu Thr Ile Ala Glu Ser Thr Thr Asp Phe Asp Met Leu 100 105 110 Thr Gly Asn His Ala Arg Asp Ala Asp Gln Phe Tyr Asp Phe Val Ala 115 120 125 Pro Met Pro Pro Ile Ala Glu Glu Phe Glu Cys Lys Ile Val Pro Val 130 135 140 Phe Ser Leu Gln Val Thr Leu Phe Pro Gly Arg Gly Ile Cys Ile Gly 145 150 155 160 Leu Ser Asn His His Cys Leu Gly Asp Ala Arg Ser Val Val Gly Phe 165 170 175 Val Leu Ala Trp Ala Ser Ile Asn Lys Phe Gly Gly Asp Glu Glu Phe 180 185 190 Leu Ser Glu Asn Gly Glu Ser Leu Pro Ile Phe Asp Arg Ser Leu Ile 195 200 205 Lys Asp Pro Leu Glu Ile Asp Thr Ile Phe Trp Lys Val Leu Arg Asn 210 215 220 Ile Pro Leu Lys Pro Ser Ser Phe Pro Leu Pro Thr Asn Arg Val Arg 225 230 235 240 Ala Thr Phe Val Leu Ser Gln Ser Asp Ile Lys Arg Leu Lys His Leu 245 250 255 Ala Asn Asn Asn Leu Val Gln Pro Ser Ser Phe Val Val Ala Ala Ala 260 265 270 Tyr Ile Trp Ser Cys Met Val Lys Ser Gly Asp Gly Gly Glu Ala Asn 275 280 285 Ala Pro Glu Leu Phe Val Ile Pro Ala Asp Ala Arg Gly Arg Thr Asn 290 295 300 Pro Pro Val Pro Ala Asn Tyr Phe Gly Asn Cys Ile Val Gly Gly Val 305 310 315 320 Val Lys Val Glu His Glu Lys Met Ala Gly Asn Glu Gly Phe Val Ile 325 330 335 Ala Ala Glu Ala Ile Ala Gly Glu Ile Lys Asn Lys Met Asn Asp Lys 340 345 350 Glu Glu Ile Leu Lys Gly Ala Glu Asn Trp Leu Ser Glu Ile Trp Lys 355 360 365 Cys Met Gly Met Ser Val Leu Gly Ile Ser Gly Ser Pro Lys Phe Asp 370 375 380 Leu Ser Asn Ala Asp Phe Gly Trp Gly Lys Ala Arg Lys Leu Glu Val 385 390 395 400 Val Ser Ile Asp Gly Glu Lys Tyr Thr Met Ser Leu Cys Asn Ser Asp 405 410 415 Cys Gly Leu Glu Val Gly Leu Ser Leu Pro Gly Glu Arg Met Glu Ala 420 425 430 Phe Ala Ala Ile Phe Ala Asp Gly Leu Ala Lys Leu Asp Ser Ser 435 440 445 28 1502 DNA Lavendula angustifolia 28 ggc acg aga att aga acc gcc atg act acc acc gtg att gaa acc acg 48 Gly Thr Arg Ile Arg Thr Ala Met Thr Thr Thr Val Ile Glu Thr Thr 1 5 10 15 gga gtc cca cct ccg ccg ggc gcc gcc gcg gag cta acg gtg cca ctc 96 Gly Val Pro Pro Pro Pro Gly Ala Ala Ala Glu Leu Thr Val Pro Leu 20 25 30 tgt ttc atg gac ttc gtt tgg ctt cat ttc cac ccc atc cgc cgc ctt 144 Cys Phe Met Asp Phe Val Trp Leu His Phe His Pro Ile Arg Arg Leu 35 40 45 att ttc tac gac cac cct tgc tct gaa tcc gac ttc cta aac gac gtc 192 Ile Phe Tyr Asp His Pro Cys Ser Glu Ser Asp Phe Leu Asn Asp Val 50 55 60 gtt ccg aag ctc aaa cac tca ctc tct ctc gct cta cag aac tat ctc 240 Val Pro Lys Leu Lys His Ser Leu Ser Leu Ala Leu Gln Asn Tyr Leu 65 70 75 80 ccg gta gct gca aac tta ctc tac cct tca gat tta aac aca gac gag 288 Pro Val Ala Ala Asn Leu Leu Tyr Pro Ser Asp Leu Asn Thr Asp Glu 85 90 95 aag ccc cta atc cgt tac gtc tcc ggc gat ggg gtt ccg ctc acc gtc 336 Lys Pro Leu Ile Arg Tyr Val Ser Gly Asp Gly Val Pro Leu Thr Val 100 105 110 gcc gtc tca gcc gcc gac ttc gac gag ctc acc gga ttt cac gtg aag 384 Ala Val Ser Ala Ala Asp Phe Asp Glu Leu Thr Gly Phe His Val Lys 115 120 125 gaa tca gat caa ttt tac gat ttc atg ccg gag atg ccg ccg gtg agg 432 Glu Ser Asp Gln Phe Tyr Asp Phe Met Pro Glu Met Pro Pro Val Arg 130 135 140 gag gaa gct ggg tgc aat tac aaa att atc cct ctc atc gcc gtg cag 480 Glu Glu Ala Gly Cys Asn Tyr Lys Ile Ile Pro Leu Ile Ala Val Gln 145 150 155 160 gtg act ctc ttc ccc ggc cgc ggg att tgc gtc ggt tta tcc aac cac 528 Val Thr Leu Phe Pro Gly Arg Gly Ile Cys Val Gly Leu Ser Asn His 165 170 175 cac tgc ctc ggc gac gcc aga tcc gtc gtc ggg ttc atg tgg cgg tgg 576 His Cys Leu Gly Asp Ala Arg Ser Val Val Gly Phe Met Trp Arg Trp 180 185 190 gcg gag atc aac aaa tcc ggc ggg gac gag gat tct caa tcg caa aac 624 Ala Glu Ile Asn Lys Ser Gly Gly Asp Glu Asp Ser Gln Ser Gln Asn 195 200 205 ggc gag tcg ttg ccg ctt ttc gat cga tcg gtt ttc gga gat cgt gat 672 Gly Glu Ser Leu Pro Leu Phe Asp Arg Ser Val Phe Gly Asp Arg Asp 210 215 220 aaa gtt aac aat atg ttt tgg gac gcg atg aag agg aaa ccg ttc gaa 720 Lys Val Asn Asn Met Phe Trp Asp Ala Met Lys Arg Lys Pro Phe Glu 225 230 235 240 gcg gcg tcg ttt ccg tta ccg acg aac aga gtg aga gga gcg ttc agc 768 Ala Ala Ser Phe Pro Leu Pro Thr Asn Arg Val Arg Gly Ala Phe Ser 245 250 255 ctc gac cca tcc gcc att aaa aag ctt aag aac cga gtt ttg tcc agt 816 Leu Asp Pro Ser Ala Ile Lys Lys Leu Lys Asn Arg Val Leu Ser Ser 260 265 270 aac caa acc cta tcc cac gtc tcc tcc ttc gtc gtg acg gct gcg tac 864 Asn Gln Thr Leu Ser His Val Ser Ser Phe Val Val Thr Ala Ala Tyr 275 280 285 gtc tgg acc tcc gtg gtg aaa tcc gcc gac gcc gcc gga gag gaa gtc 912 Val Trp Thr Ser Val Val Lys Ser Ala Asp Ala Ala Gly Glu Glu Val 290 295 300 gcc gga gac gaa gcc gat att ttc ttc ttt ccg gcc gac ggc agg ggc 960 Ala Gly Asp Glu Ala Asp Ile Phe Phe Phe Pro Ala Asp Gly Arg Gly 305 310 315 320 cgg ccg aac gcc atg gtt gac caa ccg gtg cca ctt aac tac ttc gga 1008 Arg Pro Asn Ala Met Val Asp Gln Pro Val Pro Leu Asn Tyr Phe Gly 325 330 335 aac ttt ttg ggc ggc ggg atg gtc aag atg gag cat aaa aag gtg gcg 1056 Asn Phe Leu Gly Gly Gly Met Val Lys Met Glu His Lys Lys Val Ala 340 345 350 gcg gag gac ggg ttt ctc gcg gtg gcg gag gcc atc tcc gat caa att 1104 Ala Glu Asp Gly Phe Leu Ala Val Ala Glu Ala Ile Ser Asp Gln Ile 355 360 365 aag aac aat atc aac aat aaa gaa gtt ttc atg aaa ggt acg gaa aat 1152 Lys Asn Asn Ile Asn Asn Lys Glu Val Phe Met Lys Gly Thr Glu Asn 370 375 380 tgg ttg tcg gaa atg gcg aaa gtt cct atg atg aga tca ttt gga gtt 1200 Trp Leu Ser Glu Met Ala Lys Val Pro Met Met Arg Ser Phe Gly Val 385 390 395 400 tct ggt tcg ccc aaa ttc gat ttg tcg aaa gcc gat ttc gga tgg ggg 1248 Ser Gly Ser Pro Lys Phe Asp Leu Ser Lys Ala Asp Phe Gly Trp Gly 405 410 415 aag gcg aga agg ctt gaa gtt ttg tcg atg gac gga gag aag tat tca 1296 Lys Ala Arg Arg Leu Glu Val Leu Ser Met Asp Gly Glu Lys Tyr Ser 420 425 430 atg tcg ttg tgt aat tca tca agt agc gac ggt gga tta gtc gtc gga 1344 Met Ser Leu Cys Asn Ser Ser Ser Ser Asp Gly Gly Leu Val Val Gly 435 440 445 gtt tcg ttg ccg gcg gta aga atg gag gct ttt gct tct ata ttt gaa 1392 Val Ser Leu Pro Ala Val Arg Met Glu Ala Phe Ala Ser Ile Phe Glu 450 455 460 gat ggg tta aaa tct taaattccgt tatttcgtta cttgcacaag ttcaaactat 1447 Asp Gly Leu Lys Ser 465 469 ttcatgaata aaattacttc gatttgaaca aaaaaaaaaa aaaaaaaaaa aaaaa 1502 29 469 PRT Lavendula angustifolia 29 Gly Thr Arg Ile Arg Thr Ala Met Thr Thr Thr Val Ile Glu Thr Thr 1 5 10 15 Gly Val Pro Pro Pro Pro Gly Ala Ala Ala Glu Leu Thr Val Pro Leu 20 25 30 Cys Phe Met Asp Phe Val Trp Leu His Phe His Pro Ile Arg Arg Leu 35 40 45 Ile Phe Tyr Asp His Pro Cys Ser Glu Ser Asp Phe Leu Asn Asp Val 50 55 60 Val Pro Lys Leu Lys His Ser Leu Ser Leu Ala Leu Gln Asn Tyr Leu 65 70 75 80 Pro Val Ala Ala Asn Leu Leu Tyr Pro Ser Asp Leu Asn Thr Asp Glu 85 90 95 Lys Pro Leu Ile Arg Tyr Val Ser Gly Asp Gly Val Pro Leu Thr Val 100 105 110 Ala Val Ser Ala Ala Asp Phe Asp Glu Leu Thr Gly Phe His Val Lys 115 120 125 Glu Ser Asp Gln Phe Tyr Asp Phe Met Pro Glu Met Pro Pro Val Arg 130 135 140 Glu Glu Ala Gly Cys Asn Tyr Lys Ile Ile Pro Leu Ile Ala Val Gln 145 150 155 160 Val Thr Leu Phe Pro Gly Arg Gly Ile Cys Val Gly Leu Ser Asn His 165 170 175 His Cys Leu Gly Asp Ala Arg Ser Val Val Gly Phe Met Trp Arg Trp 180 185 190 Ala Glu Ile Asn Lys Ser Gly Gly Asp Glu Asp Ser Gln Ser Gln Asn 195 200 205 Gly Glu Ser Leu Pro Leu Phe Asp Arg Ser Val Phe Gly Asp Arg Asp 210 215 220 Lys Val Asn Asn Met Phe Trp Asp Ala Met Lys Arg Lys Pro Phe Glu 225 230 235 240 Ala Ala Ser Phe Pro Leu Pro Thr Asn Arg Val Arg Gly Ala Phe Ser 245 250 255 Leu Asp Pro Ser Ala Ile Lys Lys Leu Lys Asn Arg Val Leu Ser Ser 260 265 270 Asn Gln Thr Leu Ser His Val Ser Ser Phe Val Val Thr Ala Ala Tyr 275 280 285 Val Trp Thr Ser Val Val Lys Ser Ala Asp Ala Ala Gly Glu Glu Val 290 295 300 Ala Gly Asp Glu Ala Asp Ile Phe Phe Phe Pro Ala Asp Gly Arg Gly 305 310 315 320 Arg Pro Asn Ala Met Val Asp Gln Pro Val Pro Leu Asn Tyr Phe Gly 325 330 335 Asn Phe Leu Gly Gly Gly Met Val Lys Met Glu His Lys Lys Val Ala 340 345 350 Ala Glu Asp Gly Phe Leu Ala Val Ala Glu Ala Ile Ser Asp Gln Ile 355 360 365 Lys Asn Asn Ile Asn Asn Lys Glu Val Phe Met Lys Gly Thr Glu Asn 370 375 380 Trp Leu Ser Glu Met Ala Lys Val Pro Met Met Arg Ser Phe Gly Val 385 390 395 400 Ser Gly Ser Pro Lys Phe Asp Leu Ser Lys Ala Asp Phe Gly Trp Gly 405 410 415 Lys Ala Arg Arg Leu Glu Val Leu Ser Met Asp Gly Glu Lys Tyr Ser 420 425 430 Met Ser Leu Cys Asn Ser Ser Ser Ser Asp Gly Gly Leu Val Val Gly 435 440 445 Val Ser Leu Pro Ala Val Arg Met Glu Ala Phe Ala Ser Ile Phe Glu 450 455 460 Asp Gly Leu Lys Ser 465 469 30 35 DNA Artificial sequence Description of Artificial SequencePrimer No.1 30 ggatccatcg agggacgcat gacaacaaca acaac 35 31 33 DNA Artificial sequence Description of Artificial SequencePrimer No.2 31 ggatccttac aatggttcga cgagcgccgg aga 33 32 28 DNA Artificial sequence Description of Artificial SequencePrimer No.3 32 ggacccgccg ataccggaaa attacttc 28 

1. A gene encoding a protein which has an activity to transfer an aliphatic acyl group to a glycosyl group at the 5-position of flavonoids.
 2. A gene according to claim 1, wherein the gene encodes a protein which has an amino acid sequence shown by SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29, and has an activity to transfer an aliphatic acyl group to a glycosyl group at the 5-position of flavonoids, or wherein the gene encodes a protein which has any of these amino acid sequences modified by addition or deletion of one or more amino acids and/or by substitution by other amino acids, and has an activity to transfer an aliphatic acyl group to a glycosyl group at the 5-position of flavonoids.
 3. A gene according to claim 1 or 2, wherein the gene encodes a protein which has an amino acid sequence exhibiting homology of 50% or more with an amino acid sequence shown by SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29, and which has an activity to transfer an aliphatic acyl group to a glycosyl group at the 5-position of flavonoids.
 4. A gene according to any one of claims 1 to 3, wherein the gene hybridizes with a part or all of the nucleotide sequence which encodes an amino acid sequence shown by SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29, under the condition of 5×SSC and 50° C., and wherein the gene encodes a protein which has an activity to transfer an aliphatic acyl group to a glycosyl group at the 5-position of flavonoids.
 5. A vector comprising a gene according to any one of claims 1 to
 4. 6. A host transformed by a vector according to claim
 5. 7. A protein encoding by a gene according to any one of claims 1 to
 4. 8. A process for production of a protein, comprising the steps of: culturing or growing a host according to claim 6; and collecting from the host a protein which has an activity to transfer an aliphatic acyl group to a glycosyl group at the 5-position of flavonoids.
 9. A transgenic plant having a gene according to any one of claims 1 to 4 introduced therein, or offspring or tissue thereof having the same properties.
 10. A cut flower of the transgenic plant according to claim 9 or the offspring thereof having the same properties.
 11. A method for altering the colour of flowers using a gene according to any one of claims 1 to
 4. 12. A method for blueing the colour of flowers using a gene according to any one of claims 1 to
 4. 